A. PENDAHULUAN Pulsed Field Gel Electrophoresis (PFGE) adalah metode yang digunakan untuk memisahkan DNA yang berukuran besar. Metode ini pertama kali diperkenalkan pada tahun 1984 oleh Schwartz dan Cantor. Metode PFGE ini berbeda dengan elektroforesis agarosa konvensional karena orientasi dari medan elektrik memiliki periode dengan teratur. Keberagaman medan listrik inilah yang menyebabkan DNA yang berukuran besar dapat terpisah. Prinsip dari PFGE ini adalah dengan adanya pergantian voltase di setiap elektroda tersebut, maka DNA yang berukuran besar dapat terpisah dimana fragmen DNA yang berukuran kecil akan berjalan terlebih dahulu karena memiliki waktu orientasi yang lebih sedikit dibandingkan dengan fragmen DNA yang berukuran besar yang membutuhkan waktu orientasi yang lebih lama. Teknik PFGE ini dapat digunakan untuk menganalisis kekerabatan di antara galur bakteri yang berbeda namun masih dalam satu spesies, dan menjadi metode yang sangat efektif dalam mengestimasi ukuran genom serta mengkonstruksi peta restriksi dari suatu spesies. Memanipulasi DNA dan analisis sangat mendasar dalam bidang biologi molekuler. Memang, memisahkan campuran kompleks DNA menjadi fragmen-fragmen ukuran yang berbeda oleh elektroforesis adalah teknik yang teknik yang dianggap baik pada 1970-an. Biasanya, DNA diisolasi utuh dan kemudian diberikan enzim restriksi untuk menghasilkan potongan-potongan kecil yang cukup untuk menyelesaikan melalui elektroforesis dalam agarosa atau akrilamida. Meskipun prosedur ini masih membentuk inti dari pemisahan DNA dan analisis di laboratorium saat ini telah mengalami perubahan.

Pada tahun 1984, Schwartz dan Cantor memperkenalkan cara baru untuk memisahkan DNA yakni dengan cara PFGE. Secara khusus, PFGE dapat menganalisis DNA yang sangat besar untuk pertama kalinya, menaikkan batas ukuran pemisahan DNA pada agarose 30-50 kb ke lebih dari 10 Mb (10.000 kb).Hal ini dipublikasikan dan diinformasikan sebagail instrumentasi baru dan lebih baik daripada metode yang lain.

B. Aplikasi Aplikasi PFGE sangat luas dan beragam, diantaranya kloning DNA tanaman tingkat tinggi dengan menggunakan kromosom ragi buatan, vektor kloning P1, mengidentifikasi fragmen restriksi polimorfisme panjang, pembangunan peta fisik; mendeteksi kerusakan kromosom in vivo dan degradasi, dan menentukan jumlah dan ukuran kromosom ragi, jamur, dan parasit seperti Leishmania, Plasmodium, dan Trypanosoma.

C. Teori Meskipun teori elektroforesis medan berdenyut adalah menjadi bahan perdebatan, pemeriksaan kualitatif dapat dibuat mengenai pergerakan DNA dalam gel agarosa selama PFGE. Selama PFGE kontinyu, DNA di atas 30-50 kb bermigrasi dengan mobilitas yang sama tanpa melihat ukurannya. Ini terlihat dalam gel sebagai pita difusi tunggal yang besar. Namun, jika DNA dipaksa untuk mengubah arah selama elektroforesis, ukuran fragmen yang berbeda dalam pita ini menyebar mulai terpisah satu sama lain. Setiap reorientasi dari medan listrik relatif terhadap gel, DNA berukuran lebih kecil akan mulai bergerak ke arah baru lebih cepat daripada DNA yang lebih besar. Dengan demikian, DNA yang lebih besar tertinggal, terpisah dengan DNA yang lebih kecil. Saat ini, ada tiga model yang dicoba untuk menggambarkan perilaku DNA setelah PFGE, yang dikaji oleh Chu 1990, yaitu instrumentasi model reptation bias (BRM), model rantai, dan model tas (Chu, 1990, 1991 ).

D. InstrumentasiMeskipun banyak jenis instrumentasi PFGE yang tersedia, namun hanya dikategorikan menjadi dua saja. Peralatan yang paling sederhana adalah dirancang untuk elektroforesis gel inversi medan (FIGE) (Carle, et al, 1986.). FIGE bekerja dengan berkala membalik polaritas elektroda selama elektroforesis. Karena DNA subjek FIGE ke reorientasi 180o, DNA menghabiskan waktu tertentu bergerak mundur. Hanya medan listrik switching modul diperlukan; kotak gel standar vertikal atau horizontal yang memiliki kontrol suhu bisa digunakan untuk menjalankan gel.

Meskipun lebih kompleks dalam pendekatan, nol elektroforesis lapangan terpadu (ZIFE) (Turmel, et al, 1990.) Juga jatuh ke dalam kategori pertama. Dibandingkan dengan FIGE sederhana, ZIFE sangat lambat. Namun, ZIFE mampu menyelesaikan DNA lebih besar dan memberikan porsi yang bermanfaat lebih besar dari gel. Kategori lain berisi instrumen yang reorientasi DNA pada sudut miring yang lebih kecil, umumnya antara 96 dan 120. Hal ini menyebabkan DNA untuk selalu bergerak maju dalam pola zigzag ke gel. Untuk berbagai ukuran yang sama dalam kondisi yang optimal, pemisahan ini lebih cepat, menyelesaikan berbagai ukuran yang lebih luas, dan memberikan manfaat yang lebih besar dari gel dibandingkan dengan FIGE. Gambar 1: Elektrode konfigurasi digunakan PFGE. Contour-menjepit medan listrik homogen (CHEF) (Chu, et al, 1986, 1990.);

Bidang bolak melintang elektroforesis (TAFE) dan relatif ST / RIDEtm (Stratagene);. dan gel elektroforesis berputar (RGE) merupakan contoh teknik reorientasi sudut melintang yang umum diugunakan. Dalam elaborasi lebih lanjut dari prosedur di atas, Lai dan rekan-rekannya mengembangkan elektroforesis mandiri dikendalikan Programmable (PACE) unit yang memungkinkan kontrol penuh atas sudut reorientasi, tegangan, dan waktu switch.

Berbeda dengan FIGE, sistem ini membutuhkan kotak gel khusus dengan elektroda spesifik dan geometri gel, dan kontrol elektronik untuk switching dan pemrogram mennjalankan elektroforesis. Idealnya, DNA harus terpisah di jalur lurus untuk menyederhanakan perbandingan jalur-ke-jalur. Sistem ini menggunakan medan listrik homogen yang tidak menghasilkan jalur lurus, membuat interpretasi gel sulit (Schwartz dan Cantor, 1984). Pendekatan yang sederhana untuk jalur lurus FIGE, yang menggunakan elektroda sejajar dengan medan listrik yang homogen. Meskipun sangat berguna untuk memisahkan DNA yang relatif kecil, 4 - 1.000 kb (gbr. 2), reorientasi sudut 180o FIGE tentang hasil dalam berbagai pemisahan yang paling berguna di bawah 2.000 kb. Selanjutnya, seperti teknik PFGE lain, FIGE telah inversi mobilitas di mana DNA yang lebih besar ke inverse DNA yang lebih kecil selama elektroforesis. Gambar 2. Peningkatan pemisahan kisaran 20-50 kb dengan elektroforesis gel inversi medan (FIGE). Jalankan kondisi: 230 V, 7,9 V / cm, 16 jam, 50 msec.. pulsa, maju: pulsa rasio reverse = 2,5:1, 1 GTG% agarose, TBE 0.5x, 10 Ca) 1 tangga kb, 0,5-12 kb; b) Lambda / Hind III, 0,5-23 kb, dan c) Berat molekuler, 8,3-48,5 kb.Ramping, dimana panjang pulsa reorientasi terus meningkat selama pemisahan, akan meminimalkan inversi. Kemampuan ini termasuk dalam instrumentasi yang paling akurat. Peningkatan kedua rentang pemisahan dan resolusi DNA besar membutuhkan reorientasi sudut kecil, umumnya 96-140, dengan 120 paling umum. sudut kecil (misalnya, 100) meningkatkan mobilitas DNA umumnya kecil pengaruhnya terhadap resolusi. Batas bawah adalah sekitar 96. Di bawah ini, pemisahan yang dapat dilihat

TAFE dan ST / RIDEtm menggunakan geometri yang rumit antara elektroda dan vertikal ditempatkan gel untuk mendapatkan jalur lurus. CHEF dan RGE mempertahankan medan listrik homogen dalam kombinasi dengan gel horisontal. CHEF perubahan arah medan listrik elektronik ke reorientasi DNA dengan mengubah polaritas array elektroda. Dengan RGE medan listrik adalah tetap, untuk memindahkan DNA ke arah yang baru, gel cukup berputar.

Rotating Elektroforesis Gel (RGE)

RGE adalah salah satu alat terbaru dari peralatan PFGE yang menggabungkan variabel sudut dengan medan listrik homogen (Gambar 3 dan 4) Elektroda ditempatkan di sepanjang sisi yang berlawanan dari ruang buffer dengan polaritas mereka tetap. Secara singkat, gel di tempatkan pada piring berjalan melingkar dan kemudian ditempatkan di ruang buffer. gel ini digabungkan ke drive magnetik di bawah ruang buffer untuk menghilangkan kemungkinan kebocoran yang disebabkan hubungan langsung. Untuk memaksa DNA bermigrasi ke arah baru, drive magnetik hanya memutar gel ke sudut baru. Karena sudut reorientasi DNA ditentukan oleh kopling mekanik secara langsung, RGE menawarkan banyak kelebihan termasuk biaya kecil. Tegangan, sudut, dan waktu pulsa bervariasi dengan program yang tersimpan dalam memori unit.

E. Preparasi Sampel Seiring dengan kemampuan untuk memisahkan DNA besar, diperlukan persiapan sampel baru dan prosedur penanganan. DNA besar (misal, kromosom ragi) adalah mudah dipotong dan sulit dipipet karena viskositasnya tinggi. Solusinya adalah pertama-tama menanamkan bakteri atau ragi dalam plugs agarose dan kemudian menambahkan plugs dengan enzim untuk memprose dinding sel dan protein, sehingga membuat DNA terbuka tidak rusak di agarose tersebut. Plugs kemudian dipotong sesuai ukuran, diperlakukan dengan enzim restriksi jika perlu, dimuat dalam sampel dengan baik, dan disegel ke tempatnya dengan agarose.

F. Parameter Pemisahan Beberapa parameter yang berpengaruh selama PFGE berlangsung . Ini akan dibahas secara singkat di bawah ini yang terkait dengan instrumen lapangan melintang seperti RGE. Jumlah minimum informasi yang dibutuhkan untuk mengulang pemisahan harus mencakup deskripsi singkat tentang instrumentasi lapangan berdenyut yang digunakan; tegangan diterapkan dan kekuatan yang mempengaruhi medan (misalnya, 180 V pada 5,3 / cm V), panjang pulsa (misalnya, 87 detik); sudut reorientasi (misalnya, 120), penyangga (0.5x TBE); jenis dan konsentrasi agarosa (SeaKem Emas, 1,1%); ruang buffer suhu (misalnya, 10o), jenis standar (Clontech S. cerevisiae) dan, jika mungkin, jumlah DNA yang dimuat. Meskipun data yang tercantum di atas perlu selalu mereproduksipemisahan..

Gambar 3. Rotating elektroforesis gel (RGE) pemisahan cerevisiae kromosom cercevisiae (245-2190 kb). Jalankan kondisi: 180 V, 5,1 V / cm, 34 jam, 120 sudut, 60-120 detik.. pulsa jalan, 0.5x TBE, 1,2 GTG% agarose, 10 C. Dua sisi yang digunakan gel yang sama untuk menganalisis 32 sampel dari 72 sampel.

G. Pemisahan Area Kebanyakan sistem PFGE memisahkan DNA di daerah yang relatif kecil, membatasi resolusi sampel yang kompleks. RGE adalah pengecualian untuk ini, dengan jarak pemisahan sampai 20 cm dan ukuran gel maksimum 18 x 20 cm.

H. Kekuatan Medan Kekuatan medan memiliki efek mendalam pada bidang perpisahan berdenyut dan merupakan titik temu antara waktu pemisahan dan resolusi pada tingkat tertentu. Empat sampai enam volt / cm umumnya digunakan untuk menyelesaikan DNA sampai 2000 kb (misal, kromosom S. cerevisiae) dalam jangka waktu yang normal (misalnya, 1-2 hari). Namun, kekuatan medan dapat menangkap DNA yang lebih besar dalam agarosa, dan DNA> 3000 kb memerlukan 2 V / cm akan tetapi kurang sempurna pemisahannya.

I. Waktu Bunyi Perubahan waktu bunyi terjadi terutama pada berbagai ukuran pemisahan. Bunyi kali lama menyebabkan pemisahan DNA yang lebih besar. Sebagai contoh, sebesar 5,4 / cm V, 1,6 Mb dan 2,2 Mb dari kromosom S. cerevisiae terpisah sebagai pita tunggal dengan lama bunyi 90 detik. Meningkatkan panjang bunyi menjadi 120 detik dan sudah menyelesaikan dua pita.

J. Buffer Dua buffer biasanya digunakan untuk PFGE - TAE dan TBE (1x TAE adalah 40 mM Tris asetat, 1 mM EDTA, pH 8,0; TBE 1x adalah 89 mM Tris, 89 mM asam borat, 2 mM EDTA, pH 8,0). Keduanya digunakan pada ion yang relatif rendah untuk mencegah pemanasan dan buffer 0,25 dan 0.5x untuk menunjukkan pengenceran relatif terhadap standar konsentrasi. Keuntungan penambahan larutan buffer rendah terhadap ion rendah adalah peningkatan mobilitas DNA. Misalnya, ketika menggunakan RGE untuk membandingkan buffer dan agaroses, menemukan bahwa penurunan baik TAE dan TBE menjadi 0,25 x memberikan mobilitas maksimum (40-50% lebih cepat dari 1x). 0.25x bawah, mobilitas DNA turun.

L. Agarosa Jenis pemisahan agarosa juga mempengaruhi DNA, dengan mobilitas tercepat dan resolusi terbaik dicapai dalam gel yang terbuat dari electroendosmosis rendah (EEO) agarosa. Meskipun nilai elektroforesis agarosa paling standar cocok untuk PFGE, agarosa dengan EEO minimal akan memberikan pemisahan lebih cepat. EEO rendah Beberapa "nilai field berdenyut" yang tersedia, termasuk FastLane dan Gold (FMC Bioproducts),dan Megarose (Clontech).

Gambar 4. Rotating elektroforesis gel (RGE) pemisahan 3000 hingga 6000 kb DNA kromosom pombe Schizosaccharomyces. Jalankan kondisi: 50 V, 1,4 V / cm, 100 jam, 100 sudut, menjalankan beberapa concatamated: 2500 sec./50hrs, 3000 sec./50hrs, 0.5x TBE, 0,8% megarose (Clontech), 10 C.Konsentrasi agarosa mempengaruhi baik resolusi dan mobilitas DNA (Birren, et al, 1989;. Dan White, 1992). konsentrasi yang lebih tinggi agarosa hasil lebih tajam, tetapi lebih lambat band bergerak. Dan konsentrasi biasa digunakan (0,8-1,2%) merupakan tradeoff antara kecepatan dan resolusi. persentase Tinggi agarosa EEO rendah dapat meningkatkan resolusi tanpa mengorbankan kecepatan pemisahan (White, 1992).SimakBaca secara fonetikKamus

M. Suhu

Mobilitas DNA juga tergantung pada suhu pemisahan, suhu harus konstan selama berjalan. Walaupun mobilitas tinggi meningkatkan suhu DNA, ia melakukannya dengan mengorbankan resolusi

N. Kesimpulan Sejak diperkenalkan lebih dari 8 tahun yang lalu, PFGE telah berkembang menjadi cara dan sangat bermanfaat bagi ahli biologi molekuler. Hal ini tercermin dalam ketersediaan metode secara manual. Bagaimana Masa Depan? Kemungkinan termasuk menggunakan bahan pemisahan baru atau yang ditingkatkan, dan melampaui batas ukuran saat @ 10 Mb. Laporan anekdotal menunjukkan pemisahan dalam kisaran 20 Mb atau mungkin lebih besar yang, yang selanjutnya akan menyederhanakan pekerjaan rumit tentang pemetaan genom.

DAFTAR PUSTAKA

Anand, R., and Southern, E. M. (1990). Pulsed field gel electrophoresis. In Gel Electrophoresis of Nucleic Acids: A Practical Approach. (D. Rickwood and B.D. Hames, eds.), pp. 101- 123. IRL Press at Oxford University Press, New York.Birren, B., and Lai, E. (1993). Pulsed field electrophoresis: A practical guide. Academic Press, San Diego. Birren, B., and Lai, E., eds. (1990). "Methods: A Companion to Methods of Enzymology." Pulsed-Field Electrophoresis. Vol. 1, Number 2. Academic Press, San Diego.Birren, B., Hood, L., and Lai, E. (1989). "Pulsed field gel electrophoresis: Studies of DNA migration made with the programmable, autonomously-controlled electrode electrophoresis system." Electrophoresis 10, pp. 302-309. Birren, B.W., Lai, E., Clark, S.M., Hood, L., and Simon, M.I. (1988). "Optimized conditions for pulsed field gel electrophoretic separations of DNA." Nucleic Acids Research 16, pp. 7563-7582.Butler, R., Ogilvie, D.J., Elvin, P., Riley, J.H., Finniear, R.S., Slynn, G., Morten, J.E.N., Markham, A.F., and Anand, R. (1992). Walking, cloning, and mapping with yeast artificial chromosomes: a contig encompassing D21S13 and D21S16. Carle, G.F., Frank, M., and Olson, M.V. (1986). "Electrophoretic separation of large DNA molecules by periodic inversion of the electric field." Science 232, pp. 65-68. Chu, G., Vollrath, D., and Davis, R.W. (1986). "Separation of large DNA molecules by contour-clamped homogeneous electric fields." Science 234, 1582-1585.Chu, G. (1991). "Bag model for DNA migration during pulsed-field electrophoresis." PNAS 88, 11071-11075. Chu, G. (1990). Pulsed-field electrophoresis: theory and practice. In Methods: A Companion to Methods of Enzymology. Pulsed-Field Electrophoresis (B. Birren and E. Lai, eds.), Vol. 1, No. 2, pp. 129-142. Academic Press, San Diego. Clark, S.M., Lai, E., Birren, B.W., and Hood, L. (1988). "A novel instrument for separating large DNA molecules with pulsed homogenous electric fields." Science 241, 1203-1205. Ecker, J. (1990). PFGE and YAC analysis of the Arabidopsis genome. In Methods: A Companion to Methods of Enzymology. PulsedField Electrophoresis (B. Birren and E. Lai, eds.), Vol. 1, No. 2, pp. 186-194. Academic Press, San Diego. Elia, M.C., DeLuca, J.G., and Bradley, M.O. (1991). "Significance and measurement of DNA double strand breaks in mammalian cells." Pharmacology & Therapeutics 51, pp. 291-327. Gardiner, K., Laas, W., and Patterson, D.S. (1986). "Fractionation of large mammalian DNA restriction fragments using vertical pulsed-field gradient gel electrophoresis." Somatic Cell Molec. Genet. 12, pp. 185-195.Gemmill, R.M. (1991). Pulsed field gel electrophoresis. In Advances of Electrophoresis (A. Chrambach, M.J. Dunn, and B.J. Radola, eds.), Vol. 4, pp. 1-48. VCH, Weinheim, Germany. Serwer, P. and Dunn, F. J. (1990). "Rotating gels: why, how, and what." In Methods: A Companion to Methods of Enzymology. PulsedField Electrophoresis (B. Birren and E. Lai, eds.), Vol. 1, No. 2, pp. 143-150. Academic Press, San Diego. Schwartz, D.C., and Cantor, C.R. (1984). "Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis." Cell 37, pp. 67-75.Southern, E.M., Anand, R., Brown, W.R.A., and Fletcher, D.S. (1987). "A model for the separation of large DNA molecules by crossed field gel electrophoresis." Nucleic Acids Res. 15, 5925- 5943.Turmel, C., Brassard, E., Forsyth, R., Hood, K., Slater, G.W., and Noolandi, J. (1990). High-resolution zero integrated field electrophoresis of DNA. In "Electrophoresis of Large DNA Molecules:Theory and Applications" (E. Lai and B. Birren, eds), Current Communication in Cell & Molecular Biology Vol. 1, pp. 101-131. Cold Spring Harbor Laboratory Press, New York. Van Daelen, R.A.J., and Zabel, P. (1991). Preparation of high molecular weight plant DNA and analysis by pulsed-field gel electrophoresis. In Plant Molecular Biology Manual (S.B. Gelvin, R.A. Schilperoort, and D.P.S. Verma, eds.), pp. A15/1-25. Kluwer Academic Publishers, The Netherlands.White, H.W. (1992). "Rapid separation of DNA molecules by agarose gel electrophoresis: use of a new agarose matrix and a survey of running buffer effects." Biotechniques 12, pp. 574-579.

MATA KULIAH : BIOLOGI MOLEKULER LANJUTAND O S E N : PROF. dr. MUH. NASRUM MASSI, Ph.D.

PULSED FIELD GEL ELECTROPHORESIS(PFGE)

OLEH

J A N G G AP0200310024

PROGRAM STUDI S-3 ILMU KEDOKTERANFAKULTAS KEDOKTERANUNIVERSITAS HASANUDDINMAKASSAR2011

Pulsed Field Electrophoresis for Separation of Large DNAPublished in Probe Volume 2(3): Fall 1992

Barbara Joppa, Research TechnicianSamantha Li, Research TechnicianScott Cole, Research TechnicianSean Gallagher, DirectorHSI Laboratories, Hoefer Scientific InstrumentsSan Francisco, CAManipulating and analyzing DNA are fundamentals in the field of molecular biology. Indeed, separating complex mixtures of DNA into different sized fragments by electrophoresis was a well established technique by the early 1970's. Typically, DNA was isolated intact and then treated with restriction enzymes to generate pieces small enough to resolve by electrophoresis in agarose or acrylamide. Although this procedure still forms the core of DNA separation and analysis in today's laboratories, the rules of the separation have changed. In 1984, Schwartz and Cantor described pulsed field gel electrophoresis (PFGE), introducing a new way to separate DNA. In particular, PFGE resolved extremely large DNA for the first time, raising the upper size limit of DNA separation in agarose from 30-50 kb to well over 10 Mb (10,000 kb). After this initial report, a succession of papers described new and improved instrumentation and methods. As a result, routine procedures and several commercial pulsed field units are currently available. Now, instead of cloning a large number of small fragments of DNA, PFGE permits cloning and analysis of a smaller number of very large pieces of a genome. Applications Applications of PFGE are numerous and diverse (Gemmill, 1991; Birren and Lai, 1990, 1993; and Van Daelen and Zabel, 1991). These include cloning large plant DNA using yeast artificial chromosomes (YAC's) (Ecker, 1990; see also Probe, Vol. 1, No. 1/2; and Butler, et al., 1992) and P1 cloning vectors (see Probe, Vol. 1, No. 3/4); identifying restriction fragment length polymorphisms (RFLP's) and construction of physical maps; detecting in vivo chromosome breakage and degradation (Elia, et al., 1991); and determining the number and size of chromosomes ("electrophoretic karyotype") from yeasts, fungi, and parasites such as Leishmania, Plasmodium, and Trypanosoma. Theory Although the theory of pulsed field electrophoresis is a matter of debate, qualitative statements can be made about the movement of DNA in agarose gels during PFGE. During continuous field electrophoresis, DNA above 30-50 kb migrates with the same mobility regardless of size. This is seen in a gel as a single large diffuse band. If, however, the DNA is forced to change direction during electrophoresis, different sized fragments within this diffuse band begin to separate from each other. With each reorientation of the electric field relative to the gel, smaller sized DNA will begin moving in the new direction more quickly than the larger DNA. Thus, the larger DNA lags behind, providing a separation from the smaller DNA. Currently, there are three models that attempt to describe the behavior of DNA during PFGE (reviewed by Chu, 1990), the biased reptation model (BRM), the chain model, and, most recently, the bag model (Chu, 1990, 1991). Instrumentation Although many types of PFGE instrumentation are available (fig. 1), they generally fall into two categories. The simplest equipment is designed for field inversion gel electrophoresis (FIGE) (Carle, et al., 1986). FIGE works by periodically inverting the polarity of the electrodes during electrophoresis. Because FIGE subjects DNA to a 180 reorientation, the DNA spends a certain amount of time moving backwards. Only an electrical field switching module is needed; any standard vertical or horizontal gel box that has temperature control can be used to run the gel. Although more complex in its approach, zero integrated field electrophoresis (ZIFE) (Turmel, et. al, 1990) also falls into this first category. Compared with simple FIGE, ZIFE is very slow. However, ZIFE is capable of resolving larger DNA and giving a larger useful portion of the gel. The other category contains instruments that reorient the DNA at smaller oblique angle, generally between 96 and 120. This causes DNA to always move forward in a zigzag pattern down the gel. For a similar size range under optimal conditions, these separations are faster, resolve a wider size range, and give a larger useful portion of the gel compared to FIGE. Figure 1:Electrode configuration of commonlyused pulsed field gel electrophpresis units.

Contour-clamped homogeneous electric field (CHEF) (Chu, et al., 1986, 1990);

transverse alternating field electrophoresis (TAFE) (Gardiner, et al., 1986) and its relative ST/RIDEtm (Stratagene); and rotating gel electrophoresis (RGE) (Southern, et al., 1987; Anand and Southern, 1990; Gemmill, 1991; and Serwer and Dunn, 1990) are all examples of commonly used transverse angle reorientation techniques for which instrumentation is available. In a further elaboration of the above procedures, Lai and coworkers developed the programmable autonomously controlled electrophoresis (PACE) unit which allows complete control over reorientation angle, voltage, and switch time (Clark, et al., 1988; and Birren, et al., 1989). In contrast with FIGE, these systems require both a special gel box with a specific electrode and gel geometry, and the associated electronic control for switching and programming the electrophoresis run. Ideally, the DNA should separate in straight lanes to simplify lane-to-lane comparisons. The original pulsed-field systems used inhomogeneous electric fields that did not produce straight lanes, making interpretation of gels difficult (Schwartz and Cantor, 1984). Again, the simplest approach to straight lanes is FIGE, which uses parallel electrodes to assure a homogeneous electric field. Although extremely useful for separating relatively small DNA, 4- 1,000 kb (fig. 2), FIGE's reorientation angle of 180 results in a separation range most useful under 2,000 kb. Furthermore, like other PFGE techniques, FIGE has mobility inversions in which larger DNA can move ahead of smaller DNA during electrophoresis. Figure 2. Increased separation of the 20-50 kb range with field inversion gel electrophoresis (FIGE). Run conditions: 230 V, 7.9 V/cm, 16 hrs., 50 msec. pulse, forward:reverse pulse ratio = 2.5:1, 1% GTG agarose, 0.5X TBE, 10 C.a) 1 kb ladder, 0.5-12 kb; b) Lambda/Hind III, 0.5-23 kb; and c) High molecular weight markers, 8.3-48.5 kb. Ramping, where the reorientation pulse length is constantly increased during separation, will minimize inversions. This capability is included in most commercial instrumentation. Increasing both the separation range and the resolution of large DNA requires smaller reorientation angles, generally 96-140, with 120 most common. Smaller angles (e.g., 100) increase the mobility of the DNA generally without seriously affecting resolution. The lower limit is approximately 96. Below this, separation is seriously compromised.

TAFE and ST/RIDEtm use a complicated geometry between the electrodes and a vertically placed gel to get straight lanes. CHEF and RGE maintain a homogeneous electric field in combination with a horizontal gel. CHEF changes the direction of the electric field electronically to reorient the DNA by changing the polarity of an electrode array. With RGE the electric field is fixed; to move the DNA in a new direction, the gel simply rotates.

Rotating Gel Electrophoresis RGE is one of the most recent commercial introductions of pulsed field equipment and combines variable angles with a homogeneous electric field (figs. 3 and 4) (Southern, et al., 1987; Anand and Southern, 1990; Serwer and Dunn, 1990; and Gemmill, 1991). The electrodes are positioned along opposite sides of the buffer chamber with their polarity fixed. Briefly, the gel is cast on a circular running plate and then placed in the buffer chamber. The gel is coupled to a magnetic drive beneath the buffer chamber to eliminate the possibility of leakage that a direct connection might cause. To force the migrating DNA to a new direction, the magnetic drive simply rotates the gel to the new angle. Because the reorientation angle of the DNA is determined by a straightforward mechanical coupling, RGE offers a lot of flexibility at a reduced cost. Voltage, angle, and pulse times are varied with the program stored into memory of the unit. Sample Preparation Along with the ability to separate large DNA came the need for new sample preparation and handling procedures. Large DNA (e.g., yeast chromosomes) is easily sheared and also difficult to pipet due to its high viscosity. The solution to this problem is to first embed the bacteria or yeast in agarose plugs and then treat the plugs with enzymes to digest away the cell wall and proteins, thus leaving the naked DNA undamaged in the agarose. The plugs then are cut to size, treated with restriction enzymes if necessary, loaded in the sample well, and sealed into place with agarose. Separation Parameters Several parameters act in concert during PFGE (Southern, et al., 1987; Anand and Southern, 1990; Birren, 1989; and Gemmill, 1991). These will be discussed briefly below as they relate to transverse field instruments such as RGE. The minimum amount of information needed to repeat a separation should include a short description of the pulsed field instrumentation used; applied voltage and field strength (e.g., 180 V at 5.3 V/cm); pulse length (e.g., 87 seconds); reorientation angle (e.g., 120); the buffer (0.5X TBE); the agarose type and concentration (SeaKem Gold, 1.1%); the buffer chamber temperature (e.g., 10); the type of standards (Clontech S. cerevisiae); and, if possible, the amount of DNA loaded. Although the data listed above is necessary to faithfully reproduce a separation, the information supplied in publications is rarely this complete. Figure 3. Rotating gel electrophoresis (RGE) separation Saccharomyces cercevisiae chromosomes (245-2190 kb). Run conditions: 180 V, 5.1 V/cm, 34 hrs., 120 angle, 60-120 sec. pulse ramp, 0.5X TBE, 1.2% GTG agarose, 10 C. Two combs were used on the same gel to load

32 samples, a maximum of 72 are possible Separation Area Most PFGE systems separate DNA over a relatively small area, limiting the resolution of complex samples. RGE is an exception to this, with a useful separation distance up to 20 cm and a maximum gel size of 18 x 20 cm.Field Strength The field strength has a profound effect on pulsed field separations and is a compromise between separation time and resolution of a particular size class. Four to six volts/cm is generally required for resolving DNA up to 2000 kb (e.g., S. cerevisiae chromosomes) in a reasonable period of time (e.g., 1-2 days). However, these field strengths trap and immobilize even bigger DNA in the agarose matrix, and DNA > 3000 kb requires 2 V/cm or less for separation. Pulse Time Pulse time primarily changes the size range of separation. Longer pulse times lead to separation of larger DNA. For example, at 5.4 V/cm, the 1.6 Mb and 2.2 Mb chromosomes from S. cerevisiae separate as a single band with 90-second pulse length. Increasing the pulse length to 120 seconds resolves these into two bands (Gemmill, 1991).

Reorientation Angle Any angle between 96 and 165 produces roughly equivalent separation (Birren, et al., 1988; and Gemmill, 1991). The smaller the angle, however, the faster the DNA mobility. And for separating extremely large DNA, 96 to 105 is almost a requirement to get a good separation in the shortest possible time. Buffers Two buffers are commonly employed for PFGE--TAE and TBE (1x TAE is 40 mM Tris acetate, 1 mM EDTA, pH 8.0; 1x TBE is 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0). Both are used at a relatively low ionic strength to prevent heating and carry the designations of either 0.25 and 0.5x to indicate the dilution relative to the standard concentration. An added benefit to low ionic strength buffers is an increase in DNA mobility. For example, while using RGE to compare various buffers and agaroses, White (1992) found that lowering both TAE and TBE to 0.25 x gave the maximum mobility (40-50% faster than 1x). Below 0.25x, the DNA mobility dropped off. Agarose The type of agarose also affects DNA separation, with the fastest mobilities and best resolution achieved in gels made of low electroendosmosis (EEO) agarose (Birren, et al., 1989; and White, 1992). Although most standard electrophoresis grades of agarose are suitable for PFGE (e.g., SeaKem GTG), agarose with minimal EEO will provide a faster separation. Several low EEO "pulsed field grades" are available, including FastLane and Gold (FMC BioProducts), and Megarose (Clontech). Figure 4. Rotating gel electrophoresis (RGE) separation of 3000 to 6000 kb DNA Schizosaccharomyces pombe chromosomes. Run conditions: 50 V, 1.4 V/cm, 100 hrs, 100 angle, concatamated multiple runs: 2500 sec./50hrs, 3000 sec./50hrs, 0.5X TBE, 0.8% megarose (Clontech), 10 C. The concentration of agarose affects both the resolution and mobility of the DNA (Birren, et al., 1989; and White, 1992). Higher concentrations of agarose yield sharper, but slower moving bands. And the typical concentrations used (0.8-1.2%) represent a tradeoff between speed and resolution. High percentages of low EEO agarose may improve resolution without sacrificing the speed of separation (White, 1992).

Temperature Because DNA mobility also depends on the separation temperature, the temperature must be constant both during and between runs. Although higher temperatures increase DNA mobility, it does so at the expense of resolution (Birren, et al., 1989; and Gemmill, 1991). Conclusion Since its introduction over 8 years ago, PFGE has evolved into a routine, pragmatic technique for molecular biologists. This is reflected in the present availability of methods chapters and manuals (e.g., Birren and Lai, 1990, 1993; Anand and Southern, 1990; Van Daelen and Zabel, 1991). What does the future hold? Possibilities include using a new or improved separation material, and going beyond the current size limit of @ 10 Mb. Anecdotal reports suggest separations in the range of 20 Mb or larger are possible, which would further simplify the complex task of genome mapping. For more information on Hoefer's HulaGeltm rotating gel electrophoresis unit, contact Technical Services at Hoefer Scientific Instruments at (415) 282-2307 or 800-227-4750. References Anand, R., and Southern, E. M. (1990). Pulsed field gel electrophoresis. In Gel Electrophoresis of Nucleic Acids: A Practical Approach. (D. Rickwood and B.D. Hames, eds.), pp. 101- 123. IRL Press at Oxford University Press, New York.Birren, B., and Lai, E. (1993). Pulsed field electrophoresis: A practical guide. Academic Press, San Diego. Birren, B., and Lai, E., eds. (1990). "Methods: A Companion to Methods of Enzymology." Pulsed-Field Electrophoresis. Vol. 1, Number 2. Academic Press, San Diego.Birren, B., Hood, L., and Lai, E. (1989). "Pulsed field gel electrophoresis: Studies of DNA migration made with the programmable, autonomously-controlled electrode electrophoresis system." Electrophoresis 10, pp. 302-309. Birren, B.W., Lai, E., Clark, S.M., Hood, L., and Simon, M.I. (1988). "Optimized conditions for pulsed field gel electrophoretic separations of DNA." Nucleic Acids Research 16, pp. 7563-7582.Butler, R., Ogilvie, D.J., Elvin, P., Riley, J.H., Finniear, R.S., Slynn, G., Morten, J.E.N., Markham, A.F., and Anand, R. (1992). Walking, cloning, and mapping with yeast artificial chromosomes: a contig encompassing D21S13 and D21S16. Carle, G.F., Frank, M., and Olson, M.V. (1986). "Electrophoretic separation of large DNA molecules by periodic inversion of the electric field." Science 232, pp. 65-68. Chu, G., Vollrath, D., and Davis, R.W. (1986). "Separation of large DNA molecules by contour-clamped homogeneous electric fields." Science 234, 1582-1585.Chu, G. (1991). "Bag model for DNA migration during pulsed-field electrophoresis." PNAS 88, 11071-11075. Chu, G. (1990). Pulsed-field electrophoresis: theory and practice. In Methods: A Companion to Methods of Enzymology. Pulsed-Field Electrophoresis (B. Birren and E. Lai, eds.), Vol. 1, No. 2, pp. 129-142. Academic Press, San Diego. Clark, S.M., Lai, E., Birren, B.W., and Hood, L. (1988). "A novel instrument for separating large DNA molecules with pulsed homogenous electric fields." Science 241, 1203-1205. Ecker, J. (1990). PFGE and YAC analysis of the Arabidopsis genome. In Methods: A Companion to Methods of Enzymology. PulsedField Electrophoresis (B. Birren and E. Lai, eds.), Vol. 1, No. 2, pp. 186-194. Academic Press, San Diego. Elia, M.C., DeLuca, J.G., and Bradley, M.O. (1991). "Significance and measurement of DNA double strand breaks in mammalian cells." Pharmacology & Therapeutics 51, pp. 291-327. Gardiner, K., Laas, W., and Patterson, D.S. (1986). "Fractionation of large mammalian DNA restriction fragments using vertical pulsed-field gradient gel electrophoresis." Somatic Cell Molec. Genet. 12, pp. 185-195.Gemmill, R.M. (1991). Pulsed field gel electrophoresis. In Advances of Electrophoresis (A. Chrambach, M.J. Dunn, and B.J. Radola, eds.), Vol. 4, pp. 1-48. VCH, Weinheim, Germany. Serwer, P. and Dunn, F. J. (1990). "Rotating gels: why, how, and what." In Methods: A Companion to Methods of Enzymology. PulsedField Electrophoresis (B. Birren and E. Lai, eds.), Vol. 1, No. 2, pp. 143-150. Academic Press, San Diego. Schwartz, D.C., and Cantor, C.R. (1984). "Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis." Cell 37, pp. 67-75.Southern, E.M., Anand, R., Brown, W.R.A., and Fletcher, D.S. (1987). "A model for the separation of large DNA molecules by crossed field gel electrophoresis." Nucleic Acids Res. 15, 5925- 5943.Turmel, C., Brassard, E., Forsyth, R., Hood, K., Slater, G.W., and Noolandi, J. (1990). High-resolution zero integrated field electrophoresis of DNA. In "Electrophoresis of Large DNA Molecules:Theory and Applications" (E. Lai and B. Birren, eds), Current Communication in Cell & Molecular Biology Vol. 1, pp. 101-131. Cold Spring Harbor Laboratory Press, New York. Van Daelen, R.A.J., and Zabel, P. (1991). Preparation of high molecular weight plant DNA and analysis by pulsed-field gel electrophoresis. In Plant Molecular Biology Manual (S.B. Gelvin, R.A. Schilperoort, and D.P.S. Verma, eds.), pp. A15/1-25. Kluwer Academic Publishers, The Netherlands.White, H.W. (1992). "Rapid separation of DNA molecules by agarose gel electrophoresis: use of a new agarose matrix and a survey of running buffer effects." Biotechniques 12, pp. 574-579.

Berdenyut Lapangan Elektroforesis untuk Pemisahan DNA BesarPublished in Probe Volume 2 (3): Fall 1992________________________________________Barbara Yope, Teknisi PenelitianSamantha Li, Teknisi PenelitianScott Cole, Teknisi PenelitianSean Gallagher, DirekturLaboratorium HSI, Hoefer Instrumen IlmiahSan Francisco, CAMemanipulasi DNA dan analisa adalah fundamental dalam bidang biologi molekuler. Memang, memisahkan campuran kompleks DNA menjadi fragmen-fragmen ukuran yang berbeda oleh elektroforesis adalah teknik yang mapan pada 1970-an.Biasanya, DNA diisolasi utuh dan kemudian diobati dengan enzim restriksi untuk menghasilkan potongan-potongan kecil yang cukup untuk menyelesaikan melalui elektroforesis dalam agarosa atau akrilamida. Meskipun prosedur ini masih membentuk inti dari pemisahan DNA dan analisis di laboratorium hari ini, aturan pemisahan telah berubah.Pada tahun 1984, Schwartz dan Cantor dijelaskan berdenyut lapangan elektroforesis gel (PFGE), memperkenalkan cara baru untuk DNA terpisah. Secara khusus, PFGE diselesaikan DNA yang sangat besar untuk pertama kalinya, menaikkan batas ukuran atas pemisahan DNA pada agarose 30-50 kb ke lebih dari 10 Mb (10.000 kb).Setelah laporan awal, suksesi kertas dijelaskan instrumentasi baru dan lebih baik dan metode. Akibatnya, prosedur rutin dan beberapa unit lapangan berdenyut komersial saat ini tersedia. Sekarang, bukannya kloning sejumlah besar fragmen kecil dari DNA, PFGE izin kloning dan analisis dari sejumlah kecil potongan yang sangat besar genom.AplikasiAplikasi PFGE sangat banyak dan beragam (Gemmill, 1991; Birren dan Lai, 1990, 1993; dan Van Daelen dan Zabel, 1991). Ini termasuk kloning DNA tanaman besar dengan menggunakan kromosom ragi buatan (YAC's) (Ecker, 1990, lihat juga Probe, Vol 1, No 1 / 2,.. Dan Butler, et al, 1992) dan vektor kloning P1 (lihat Probe, Vol . 1, No 3 / 4); mengidentifikasi polimorfisme panjang fragmen restriksi (RFLP) dan pembangunan peta fisik; mendeteksi kerusakan kromosom di vivo dan degradasi (Elia, et al, 1991);. dan menentukan jumlah dan ukuran kromosom ( "elektroforetik kariotipe") dari ragi, jamur, dan parasit seperti Leishmania, Plasmodium, dan Trypanosoma.TeoriMeskipun teori elektroforesis medan berdenyut adalah menjadi bahan perdebatan, pernyataan kualitatif dapat dibuat tentang pergerakan DNA dalam gel agarosa selama PFGE. Selama lapangan elektroforesis kontinyu, DNA di atas 30-50 kb bermigrasi dengan mobilitas yang sama tanpa memandang ukurannya. Ini terlihat dalam gel sebagai band diffuse tunggal yang besar. Namun, jika DNA dipaksa untuk mengubah arah selama elektroforesis, ukuran fragmen yang berbeda dalam band ini menyebar mulai terpisah dari satu sama lain.Dengan setiap reorientasi dari medan listrik relatif terhadap gel, DNA berukuran lebih kecil akan mulai bergerak ke arah baru lebih cepat daripada DNA yang lebih besar. Dengan demikian, DNA yang lebih besar tertinggal, memberikan pemisahan dari DNA yang lebih kecil.Saat ini, ada tiga model yang mencoba untuk menggambarkan perilaku DNA selama PFGE (dikaji oleh Chu, 1990), model reptation bias (BRM), model rantai, dan, terakhir, model tas (Chu, 1990, 1991 ).InstrumentasiMeskipun banyak jenis instrumentasi PFGE tersedia (gbr. 1), mereka umumnya jatuh ke dalam dua kategori. Peralatan yang paling sederhana adalah dirancang untuk elektroforesis gel inversi medan (FIGE) (Carle, et al, 1986.). FIGE bekerja dengan berkala membalik polaritas elektroda selama elektroforesis. Karena DNA subjek FIGE ke reorientasi 180o, DNA menghabiskan waktu tertentu bergerak mundur. Hanya medan listrik switching modul diperlukan; sembarang kotak gel standar vertikal atau horizontal yang memiliki kontrol suhu bisa digunakan untuk menjalankan gel.Meskipun lebih kompleks dalam pendekatan, nol elektroforesis lapangan terpadu (ZIFE) (Turmel, et al, 1990.) Juga jatuh ke dalam kategori pertama. Dibandingkan dengan FIGE sederhana, ZIFE sangat lambat. Namun, ZIFE mampu menyelesaikan DNA lebih besar dan memberikan porsi yang bermanfaat lebih besar dari gel.Kategori lain berisi instrumen yang reorientasi DNA pada sudut miring yang lebih kecil, umumnya antara 96 dan 120. Hal ini menyebabkan DNA untuk selalu bergerak maju dalam pola zigzag ke gel. Untuk berbagai ukuran yang sama dalam kondisi yang optimal, pemisahan ini lebih cepat, menyelesaikan berbagai ukuran yang lebih luas, dan memberikan sebagian manfaat yang lebih besar dari gel dibandingkan dengan FIGE.Gambar 1: elektrode konfigurasi umumdigunakan unit gel lapangan electrophpresis berdenyut.

Contour-menjepit medan listrik homogen (CHEF) (Chu, et al, 1986, 1990.);

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melintang lapangan elektroforesis bolak (TAFE) (Gardiner, et al, 1986.) dan relatif ST / RIDEtm (Stratagene);. dan berputar elektroforesis gel (RGE) (Selatan, et al, 1987; Anand dan Selatan, 1990; Gemmill, 1991; dan Serwer dan Dunn, 1990) merupakan contoh yang umum digunakan teknik reorientasi sudut melintang yang instrumentasi tersedia. Dalam elaborasi lebih lanjut dari prosedur di atas, Lai dan rekan kerja mengembangkan elektroforesis mandiri dikendalikan Programmable (PACE) unit yang memungkinkan kontrol penuh atas sudut reorientasi, tegangan, dan waktu switch (Clark, et al, 1988;. Dan Birren, et al. , 1989). Berbeda dengan FIGE, sistem ini membutuhkan baik kotak gel khusus dengan elektroda spesifik dan geometri gel, dan kontrol elektronik terkait untuk switching dan pemrograman jalankan elektroforesis.Idealnya, DNA harus terpisah di jalur lurus untuk menyederhanakan perbandingan jalur-ke-jalur. The berdenyut-field asli sistem yang digunakan medan listrik homogen yang tidak menghasilkan jalur lurus, membuat interpretasi gel sulit (Schwartz dan Cantor, 1984). Sekali lagi, pendekatan yang sederhana untuk jalur lurus FIGE, yang menggunakan elektroda sejajar dengan menjamin medan listrik homogen.Meskipun sangat berguna untuk memisahkan DNA yang relatif kecil, 4 - 1.000 kb (gbr. 2), reorientasi sudut 180o FIGE tentang hasil dalam berbagai pemisahan yang paling berguna di bawah 2.000 kb. Selanjutnya, seperti teknik PFGE lain, FIGE telah inversi mobilitas di mana DNA yang lebih besar dapat bergerak maju DNA yang lebih kecil selama elektroforesis.Gambar 2. Peningkatan pemisahan kisaran 20-50 kb dengan elektroforesis gel inversi medan (FIGE). Jalankan kondisi: 230 V, 7,9 V / cm, 16 jam, 50 msec.. pulsa, maju: pulsa rasio reverse = 2,5:1, 1 GTG% agarose, TBE 0.5x, 10 Ca) 1 tangga kb, 0,5-12 kb; b) Lambda / Hind III, 0,5-23 kb, dan c) Tinggi molekuler berat spidol, 8,3-48,5 kb.Ramping, dimana panjang pulsa reorientasi terus meningkat selama pemisahan, akan meminimalkan inversi. Kemampuan ini termasuk dalam instrumentasi yang paling komersial.Peningkatan kedua rentang pemisahan dan resolusi DNA besar membutuhkan reorientasi sudut kecil, umumnya 96-140, dengan 120 paling umum. sudut kecil (misalnya, 100) meningkatkan mobilitas DNA umumnya tanpa serius mempengaruhi resolusi. Batas bawah adalah sekitar 96. Di bawah ini, pemisahan serius dikompromikan.

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TAFE dan ST / RIDEtm menggunakan geometri yang rumit antara elektroda dan vertikal ditempatkan gel untuk mendapatkan jalur lurus. CHEF dan RGE mempertahankan medan listrik homogen dalam kombinasi dengan gel horisontal. CHEF perubahan arah medan listrik elektronik ke reorientasi DNA dengan mengubah polaritas array elektroda. Dengan RGE medan listrik adalah tetap, untuk memindahkan DNA ke arah yang baru, gel cukup berputar.

Rotating Elektroforesis GelRGE adalah salah satu perkenalan komersial terbaru dari peralatan lapangan berdenyut dan menggabungkan variabel sudut dengan medan listrik homogen (Gambar 3 dan 4) (Selatan, et al, 1987;. Anand dan Selatan, 1990; Serwer dan Dunn, 1990; dan Gemmill, 1991). Elektroda ditempatkan di sepanjang sisi yang berlawanan dari ruang buffer dengan polaritas mereka tetap. Secara singkat, gel adalah dilemparkan pada piring berjalan melingkar dan kemudian ditempatkan di ruang buffer. gel ini digabungkan ke drive magnetik di bawah ruang buffer untuk menghilangkan kemungkinan kebocoran yang koneksi langsung dapat menyebabkan.Untuk memaksa DNA bermigrasi ke arah baru, drive magnetik hanya berputar gel ke sudut baru. Karena sudut reorientasi DNA ditentukan oleh kopling mekanik langsung, RGE menawarkan banyak fleksibilitas dengan biaya berkurang. Tegangan, sudut, dan waktu pulsa bervariasi dengan program yang tersimpan dalam memori unit.Preparasi SampelSeiring dengan kemampuan untuk memisahkan DNA besar datang kebutuhan untuk persiapan sampel baru dan prosedur penanganan. DNA besar (misal, kromosom ragi) adalah mudah dicukur dan juga sulit untuk pipet karena viskositas tinggi. Solusi untuk masalah ini adalah pertama-tama menanamkan bakteri atau ragi dalam plugs agarose dan kemudian memperlakukan plugs dengan enzim untuk mencerna menjauh dinding sel dan protein, sehingga membuat DNA telanjang tidak rusak di agarose tersebut. Plugs kemudian dipotong sesuai ukuran, diperlakukan dengan enzim restriksi jika perlu, dimuat dalam sampel dengan baik, dan disegel ke tempatnya dengan agarose.Pemisahan ParameterBeberapa parameter bertindak dalam konser selama PFGE (Selatan, et al, 1987;. Anand dan Selatan, 1990; Birren, 1989; dan Gemmill, 1991). Ini akan dibahas secara singkat di bawah ini karena terkait dengan instrumen lapangan melintang seperti RGE. Jumlah minimum informasi yang dibutuhkan untuk mengulang perpisahan harus mencakup deskripsi singkat tentang instrumentasi lapangan berdenyut digunakan; tegangan diterapkan dan kekuatan medan (misalnya, 180 V pada 5,3 / cm V), panjang pulsa (misalnya, 87 detik); sudut reorientasi (misalnya, 120), penyangga (0.5x TBE); jenis dan konsentrasi agarosa (SeaKem Emas, 1,1%); ruang buffer suhu (misalnya, 10o), jenis standar (Clontech S. cerevisiae) dan, jika mungkin, jumlah DNA yang dimuat. Meskipun data yang tercantum di atas perlu setia mereproduksi pemisahan, informasi yang diberikan dalam publikasi jarang ini selesai.Gambar 3. Rotating elektroforesis gel (RGE) pemisahan cerevisiae kromosom cercevisiae (245-2190 kb). Jalankan kondisi: 180 V, 5,1 V / cm, 34 jam, 120 sudut, 60-120 detik.. pulsa jalan, 0.5x TBE, 1,2 GTG% agarose, 10 C. Dua sisir yang digunakan pada gel yang sama untuk me-load

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32 sampel, maksimal 72 yang mungkinPemisahan AreaKebanyakan sistem PFGE terpisah DNA di daerah yang relatif kecil, membatasi resolusi sampel yang kompleks. RGE adalah pengecualian untuk ini, dengan jarak pemisahan yang berguna sampai 20 cm dan ukuran gel maksimum 18 x 20 cm.Field StrengthKekuatan medan memiliki efek mendalam pada bidang perpisahan berdenyut dan merupakan kompromi antara waktu pemisahan dan resolusi kelas ukuran tertentu. Empat sampai enam volt / cm umumnya diwajibkan untuk menyelesaikan DNA sampai dengan 2000 kb (misal, kromosom S. cerevisiae) dalam jangka waktu yang wajar (misalnya, 1-2 hari). Namun, lapangan kekuatan perangkap dan melumpuhkan DNA yang lebih besar dalam agarosa, dan DNA> 3000 kb memerlukan 2 V / cm atau kurang untuk pemisahan.Pulse WaktuPulse waktu terutama perubahan berbagai ukuran pemisahan. pulsa kali lebih lama menyebabkan pemisahan DNA yang lebih besar. Sebagai contoh, sebesar 5,4 / cm V, 1,6 Mb dan 2,2 Mb dari kromosom S. cerevisiae terpisah sebagai band tunggal dengan panjang pulsa 90 detik. Meningkatkan panjang pulsa untuk 120 detik menyelesaikan ini menjadi dua band (Gemmill, 1991).

Reorientasi AngleSetiap sudut antara 96 dan 165 menghasilkan sekitar pemisahan setara (Birren, et al, 1988;. Dan Gemmill, 1991). Semakin kecil sudut, bagaimanapun, semakin cepat mobilitas DNA. Dan untuk memisahkan DNA yang sangat besar, 96 untuk 105 hampir satu persyaratan untuk mendapatkan pemisahan yang baik dalam waktu sesingkat mungkin.BufferDua buffer biasanya digunakan untuk PFGE - TAE dan TBE (1x TAE adalah 40 mM Tris asetat, 1 mM EDTA, pH 8,0; TBE 1x adalah 89 mM Tris, 89 mM asam borat, 2 mM EDTA, pH 8,0). Keduanya digunakan pada kekuatan ion yang relatif rendah untuk mencegah pemanasan dan membawa sebutan baik 0,25 dan 0.5x untuk menunjukkan pengenceran relatif terhadap konsentrasi standar. Keuntungan ditambahkan ke buffer rendah kekuatan ion adalah peningkatan mobilitas DNA. Misalnya, ketika menggunakan RGE untuk membandingkan berbagai buffer dan agaroses, White (1992) menemukan bahwa penurunan baik TAE dan TBE menjadi 0,25 x memberikan mobilitas maksimum (40-50% lebih cepat dari 1x). 0.25x bawah, mobilitas DNA turun.AgarosaJenis pemisahan agarosa juga mempengaruhi DNA, dengan Mobilitas tercepat dan resolusi terbaik dicapai dalam gel yang terbuat dari electroendosmosis rendah (EEO) agarosa (Birren, et al, 1989;. Dan White, 1992). Meskipun nilai elektroforesis agarosa paling standar cocok untuk PFGE (misalnya, SeaKem GTG), agarosa dengan EEO minimal akan memberikan pemisahan lebih cepat. EEO rendah Beberapa "nilai field berdenyut" yang tersedia, termasuk FastLane dan Gold (FMC Bioproducts), dan Megarose (Clontech).Gambar 4. Rotating elektroforesis gel (RGE) pemisahan 3000 hingga 6000 kb DNA kromosom pombe Schizosaccharomyces. Jalankan kondisi: 50 V, 1,4 V / cm, 100 jam, 100 sudut, menjalankan beberapa concatamated: 2500 sec./50hrs, 3000 sec./50hrs, 0.5x TBE, 0,8% megarose (Clontech), 10 C.Konsentrasi agarosa mempengaruhi baik resolusi dan mobilitas DNA (Birren, et al, 1989;. Dan White, 1992). konsentrasi yang lebih tinggi agarosa hasil lebih tajam, tetapi lebih lambat band bergerak. Dan konsentrasi biasa digunakan (0,8-1,2%) merupakan tradeoff antara kecepatan dan resolusi. persentase Tinggi agarosa EEO rendah dapat meningkatkan resolusi tanpa mengorbankan kecepatan pemisahan (White, 1992).SimakBaca secara fonetikKamusGoogle Terjemahan untuk:PenelusuranVideoEmailPonsel

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SuhuKarena mobilitas DNA juga tergantung pada suhu pemisahan, suhu harus konstan baik selama dan antara berjalan. Walaupun mobilitas tinggi meningkatkan suhu DNA, ia melakukannya dengan mengorbankan resolusi (Birren, et al, 1989;. dan Gemmill, 1991).KesimpulanSejak diperkenalkan lebih dari 8 tahun yang lalu, PFGE telah berkembang menjadi teknik, rutin pragmatis bagi ahli biologi molekuler. Hal ini tercermin dalam ketersediaan kini bab metode dan manual (misalnya, Birren dan Lai, 1990, 1993; Anand dan Selatan, 1990; Van Daelen dan Zabel, 1991).Apa Masa Depan? Kemungkinan termasuk menggunakan bahan pemisahan baru atau yang ditingkatkan, dan melampaui batas ukuran saat @ 10 Mb. Laporan anekdotal menunjukkan pemisahan dalam kisaran 20 Mb atau lebih besar yang mungkin, yang selanjutnya akan menyederhanakan tugas kompleks pemetaan genom.Untuk informasi lebih lanjut tentang Hoefer's HulaGeltm berputar unit elektroforesis gel, hubungi Layanan Teknis di Hoefer Ilmiah Instrumen di (415) 282-2307 atau 800-227-4750.

SuhuKarena mobilitas DNA juga tergantung pada suhu pemisahan, suhu harus konstan baik selama dan antara berjalan. Walaupun mobilitas tinggi meningkatkan suhu DNA, ia melakukannya dengan mengorbankan resolusi (Birren, et al, 1989;. dan Gemmill, 1991).KesimpulanSejak diperkenalkan lebih dari 8 tahun yang lalu, PFGE telah berkembang menjadi teknik, rutin pragmatis bagi ahli biologi molekuler. Hal ini tercermin dalam ketersediaan kini bab metode dan manual (misalnya, Birren dan Lai, 1990, 1993; Anand dan Selatan, 1990; Van Daelen dan Zabel, 1991).Apa Masa Depan? Kemungkinan termasuk menggunakan bahan pemisahan baru atau yang ditingkatkan, dan melampaui batas ukuran saat @ 10 Mb. Laporan anekdotal menunjukkan pemisahan dalam kisaran 20 Mb atau lebih besar yang mungkin, yang selanjutnya akan menyederhanakan tugas kompleks pemetaan genom.Untuk informasi lebih lanjut tentang Hoefer's HulaGeltm berputar unit elektroforesis gel, hubungi Layanan Teknis di Hoefer Ilmiah Instrumen di (415) 282-2307 atau 800-227-4750.SuhuKarena mobilitas DNA juga tergantung pada suhu pemisahan, suhu harus konstan baik selama dan antara berjalan. Walaupun mobilitas tinggi meningkatkan suhu DNA, ia melakukannya dengan mengorbankan resolusi (Birren, et al, 1989;. dan Gemmill, 1991).KesimpulanSejak diperkenalkan lebih dari 8 tahun yang lalu, PFGE telah berkembang menjadi teknik, rutin pragmatis bagi ahli biologi molekuler. Hal ini tercermin dalam ketersediaan kini bab metode dan manual (misalnya, Birren dan Lai, 1990, 1993; Anand dan Selatan, 1990; Van Daelen dan Zabel, 1991).Apa Masa Depan? Kemungkinan termasuk menggunakan bahan pemisahan baru atau yang ditingkatkan, dan melampaui batas ukuran saat @ 10 Mb. Laporan anekdotal menunjukkan pemisahan dalam kisaran 20 Mb atau lebih besar yang mungkin, yang selanjutnya akan menyederhanakan tugas kompleks pemetaan genom.Untuk informasi lebih lanjut tentang Hoefer's HulaGeltm berputar unit elektroforesis gel, hubungi Layanan Teknis di Hoefer Ilmiah Instrumen di (415) 282-2307 atau 800-227-4750.

Pulsed-Field Gel Electrophoresis (PFGE) Techniqueand its use in Molecular BiologyEsin (HACIOLU) BASIMS.leyman Demirel University, Faculty of Agriculture, Department of Plant Protection,32260 ..n.r, Isparta - TURKEYH.seyin BASIMAkdeniz University, Faculty of Agriculture, Department of Plant Protection,07058, Antalya - TURKEYReceived: 30.06.2000Abstract: In recent years, the use of pulsed-field gel electrophoresis (PFGE) in the molecular biologyarea has been subject to much research. PFGE is a powerful tool for characterizing various strains atthe DNA level, obtaining relevant information on genome size and constructing the physical andgenetic map of the chromosome of bacteria that are poorly understood at the genetic level as well asin separating chromosomes in microorganisms, and in the long-range mapping of mammalian genes.PFGE also has advantage of examining the elongated and oriented configuration of large DNAmolecules in agarose gels at finite field strengths. In this review, the use of PFGE in molecular biology,the general characteristics of PFGE, different types of PFGE and factors affecting PFGE areintroduced.Key Words: Pulsed-Field Gel Electrophoresis (PFGE), CHEF, Molecular Biology, Biotechnology,Restriction Enzymes.Pulsed-Field Jel Elektroforez (PFGE) Teknii ve Molek.ler BiyolojiAlannda Kullanm.zet: Son yllarda, molek.ler biyoloji alannda pulsed-field jel elektroforez (PFGE)in kullanm birokaratrmaya konu olmutur. PFGE, DNA d.zeyinde eitli strainlerin karakterize edilmesinde, genomb.y.kl.kleri hakknda bilgi elde etmede, genetik d.zeyde anlalamam bakteri kromozomlarnnfiziki ve genetik haritalarnn oluturulmasnda,mikroorganizmalarn kromozomlarnn ayrlmasnda,ve memeli genlerinin b.y.k aptaki haritalanmasnda kullanlan etkili bir metottur. PFGE; agaroz jelierisindeki b.y.k DNA molek.llerinin uygun bir alanda deiik ekillerde hareket etmesini salayanbir avantaja da sahiptir. Bu derlemede, PFGEin molek.ler biyoloji alannda kullanm, PFGEin genel.zellikleri, eitli tipleri ve PFGEi etkileyen fakt.rler sunulmutur.Anahtar S.zc.kler: Pulsed-Field Jel Elektroforez (PFGE), CHEF, Molek.ler Biyoloji, Biyoteknoloji,Restriksiyon Enzimler405Turk J Biol25 (2001) 405-418 T.BTAKPulsed-Field Gel Electrophoresis (PFGE) Technique and its use in Molecular Biology406IntroductionMuch of the rapid progress that is being made in molecular biology today depends upon theability to separate, size and visualize DNA molecules. The most common technique for thispurpose is that of standard agarose gel electrophoresis. Gel electrophoresis (1) is one of themost commonly used separation techniques in the modern biology laboratory. Its ubiquityarises from both the simplicity and versatility of the technique. Electrophoresis has foundwidespread use in biological assays, and in the purification and separation of proteins andnucleic acids. The physical mapping of genes and DNA sequencing both depend on separationby gel electrophoresis (1). Conventional gel electrophoresis of DNA molecules is carried out byplacing DNA in a solid matrix (i.e. agarose or polyacrylamide) and inducing the molecules tomigrate through the gel under a static electric field. DNA fragments from 100 to 200 base pairs(bp) up to 50 kilobase pairs (kb) are routinely separated by conventional gel electrophoresistechniques. Above 50 kb, because of the size of the molecules, the sieving action of the gel islost, and fragments run as a broad, unresolved band with anomalously high mobility. Althoughlarger fragments (up to 750 kb) have been resolved by this technique (2), the gels used areextremely fragile due to the very low agarose concentrations, and the separation is notadequate for most applications. The separation of DNA molecules by other techniques is timeconsuming.The need for the analysis of large DNA molecules is in large-scale mapping (3).In 1982, Schwartz et al. (4) introduced the concept that DNA molecules larger than 50 kbcan be separated by using two alternating electric fields (i.e. PFGE). Since that time, a numberof instruments based on this principle have been developed, and the value of using pulsed fieldshas been demonstrated for separating DNAs from a few kb to over 10 megabase pairs (Mb).The development of PFGE has increased by two orders of magnitude the size of DNAmolecules that can be routinely fractionated and analyzed. This increase is of major importancein molecular biology because it simplifies many previously laborious investigations and makespossible many new ones. Its range of application spans all organisms (2) from bacteria andviruses to mammals (5). PFGE has shown excellent ability to separate small, natural linearchromosomal DNAs ranging in size from 50-kb parasite microchromosomes to multimillion-bpyeast chromosomes. However, intact human chromosomes range in size from 50 million to250 million bp (Mb), too large for direct PFGE separations (6). PFGE provides the means forthe routine separation of fragments exceeding 6,000 kb (2, 7, 8, 9). Therefore, PFGEseparates DNAs from a few kilobase (kb) to over 10 megabase pairs (Mb) (10).The new technique of PFGE takes advantage of the elongated and oriented configuration oflarge DNA molecules in agarose gels at finite field strengths. An important bonus of thistechnique is the ease with which the genome size can be measured, a parameter that waspreviously subject to considerable error when measured by other techniques. One importantoutcome of the use of PFGE and restriction endonuclease digestion is the construction of aphysical map. General applications of PFGE can be in the separation of whole chromosomes,the large - scale restriction mapping of chromosome regions and in using DNA fragmentpurification as an aid in cloning. PFGE will greatly facilitate the precise selection of very largefragments for cloning, and it provides rapid analysis of a large chromosomal region. PFGE hasproven extremely powerful in the analysis of large DNA molecules from a variety of sources,including specifically fragmented genomes of bacteria (11), mammals (5), parasite protozoa(12-15) and intact chromosomal DNAs from fungi (16-18). The introduction of PFGEtechniques for separating large DNA molecules has had an invigorating effect on the study ofchromosomal DNA molecules, genome structure and electrophoretical theory.In this review, the use of PFGE in molecular biology the general characteristics of PFGE,different types of PFGE and factors affecting PFGE are introduced.Types of PFGEPFGE size resolves DNA molecules of almost a millimeter in length through the use ofpulsed-field electric fields, which selectively modulate mobilities in a size-dependent fashion.The pulsed electrophoresis effect has been utilized by a variety of instruments (FIGE, TAFE,CHEF, OFAGE, PACE and rotating electrode gel) to increase the size resolution of both largeand small DNA molecules (1). It is important when choosing a PFGE system to evaluate costand performance in the light of projected use. There are different types of PFGE. These are:1. Field-Inversion Gel Electrophoresis (FIGE): In 1986, Carle, Frank and Olson developeda simpler system, FIGE, in which the two fields were 180 apart (19). Electrode polarity wasreversed at intervals, with a longer forward than reverse pulse time to generate a net forwardsample migration. Net forward migration is achieved by increasing the ratio of forward toreverse pulse times to 3:1. To improve the resolution of the bands by FIGE, the duration ofpulse times is increased progressively during a run. This is called switch time ramping. Bychanging pulse durations continually during the course of an experiment, FIGE has theadvantages of straight lanes and simple equipment. All that is needed are standard gel boxesand a pulse controller. Today, FIGE is very popular for smaller fragment separations. FIGEprovides acceptable resolution up to 800 kilobases (600-750 kb).2. Transverse-Alternating Field Gel Electrophoresis (TAFE): This form of PFGE allowsseparation of large DNA fragments in a simple, convenient format without the drawbacks ofearlier pulsed-field techniques. In TAFE, the gel is oriented vertically and a simple four-electrodearray is placed not in the plane of the gel, but in front and at the back of it. Sample moleculesare forced to zigzag through the thickness of the gel, and all lanes experience the same effects,so the bands remain straight (20). As the molecules move down the gel, they are subjected tocontinual variations in field strength and reorientation angle, but to all lanes equally. However,the angle between the electric fields varies from the top of the gel (115) to the bottom(approximately 165) and hence molecules still do not move at a constant velocity over the407E. (HACIOLU) BASIM, H. BASIMlength of the gel. TAFE technology, with regular and sharp separation of DNA bands, will be ofspecial advantage in the study of genetics of many pathogenic protozoans, where such analysiswas impossible before (20). TAFE has been used for the separation of fragments up to 1,600kilobase fragments.3. Contour-Clamped Homogeneous Electric Fields (CHEF): CHEF is the most widely usedapparatus. The CHEF apparatus provides a more sophisticated solution to the distorting effectsof both the edges of the chamber and the passive electrodes. CHEF has twenty-four pointelectrodes equally spaced around the hexagonal contour. In the CHEF system, there are nopassive electrodes. All the electrodes are connected to the power supply via an external loopof resistors, all of which have the same resistance. This loop is responsible for setting thevoltages of all the electrodes around the hexagonal contour to values appropriate to thegeneration of uniform fields in each of the alternate switching positions. The CHEF system setsthe voltages at these 24 points. This apparatus produces electric fields that are sufficientlyuniform so that all lanes of a gel run straight. CHEF uses an angle of reorientation of 120 withgradiations of electropotential radiating from the positive to the negative pores. Molecules upto 7,000 kb can be separated by CHEF (10).4. Orthogonal-Field Alternation Gel Electrophoresis (OFAGE): A similar apparatus thatused two nonhomogeneous electric fields was reported by Carle and Olson (17) in 1984. Themajor drawbacks of these apparatuses were that because the electric fields were not uniform,and the angle between the electric field varied across the gel, DNA molecules migrated atdifferent rates depending on their location in the gel. This is especially problematic inmammalian genome mapping, where a continuous distribution of fragment sizes is generated.Lane-to-lane comparisions and size estimations for digested genomic DNA are lessstraightforward when fewer discrete bands are being separated, as with the chromosomes oflower organisms like yeast. The angle between the electric fields varies from less than 180 andthe more than 90. DNA molecules from 1,000 to 2,000 kb can be separated in OFAGE (17,21).5. Rotating Gel Electrophoresis (RGE): In England in 1987, Southern (22) described anovel PFGE system that rotates the gel between two set angles while the electrodes are off. InRGE, the electric field is uniform and bands are straight because only one set of electrodes isused. RGE makes it easy to perform time and voltage ramping. It also enables users to studythe effects of different angles, and even to vary these, during an experiment-angle ramping.RGE uses a single homogeneous field and changes the orientation of the electric field in relationto the gel by discontinuously and periodically rotating the gel. Switch times are too long in RGE.The DNA molecules migrate in straight lanes, due to the homogeneous fields, and DNAmolecules from 50 kb to 6,000 kb can be separated by adjusting the frequency of the gelrotation. In addition, the angle of reorientation can be easily altered simply by changing theangle of rotation (2, 23).Pulsed-Field Gel Electrophoresis (PFGE) Technique and its use in Molecular Biology4086. Programmable Autonomously-Controlled Electrodes (PACE): The PACEelectrophoresis system offers precise control over all electric field parameters by theindependent regulation of the voltages on 24 electrodes arranged in a closed contour. Theflexibility of the PACE system derives from its ability to generate an unlimited number ofelectric fields of controlled homogeneity, voltage gradient, orientation and duration. The PACEsystem can perform all previous pulsed field switching regimens (i.e. FIGE, OFAGE, PHOGE,unidirectional pulsing), as well as generate voltage clamped homogeneous static fields. ThePACE system separates DNA fragments from 100 bp to over 6 Mb. The ability to alter thereorientation angle between the alternating fields permits an increased speed of separation forlarge DNA molecules. A computer-driven system known as PACE, designed by Lai et al. (1) maybe the ultimate PFGE device. It is an extremely useful tool for studying variables such as pulsetime, temperature, agarose concentration, voltage and angles between fields affecting DNAmigration in PFGE (24).7. Pulsed-Homogeneous Orthogonal Field Gel Electrophoresis (PHOGE): The majordifference between this instrument and other gel boxes with homogeneous electric fields is thatthe field reorientation angle is 90. PHOGE uses a 90 reorientation angle, but the DNAmolecules undergo four reorientations per cycle instead of two. The DNA lanes in PHOGE donot run straight, a phenomenon which has been described for gel runs involving multipleelectric fields in this manner. This system separates DNA fragments of up to 1 Mb (23).PFGE EquipmentThe basic components of a PFGE system consist of a gel box with some means oftemperature regulation, a switching unit for controlling the electric fields, a cooler and a powersupply (1).Gel BoxThe basic design of PFGE boxes consists of an immobilized gel within an array of electrodesand a means of circulating the electrophoresis buffer. Voltage gradients of 10 volts/cm arecommonly used in PFGE. Voltage gradients as high as 15 volts/cm have been used in fieldinversion separations of cosmid clones (1). The temperature of the buffer is controlled by aheat-exchange mechanism. Generally, the buffer is recirculated throughout the gel box usinginlet and outlet ports (17, 24).High Voltage Power SupplyPrecise control of the electric field gradient is necessary to obtain consistent PFGEseparations. The output ratings of the power supply should therefore be high enough to meet409E. (HACIOLU) BASIM, H. BASIMboth the voltage and current requirements of the gel box. A typical PFGE gel box has electrodesthat are 25 to 50 cm apart. To achieve the commonly used range of voltage gradients of 1.5to 15 volts/cm requires a power supply with a maximum voltage rating of 750 volts. Thecurrent drawn at this voltage in most PFGE boxes is about 0.5 amperes at 14C using 0.5xTBE (1x TBE is 89 mM Tris pH: 7. 89 mM Boric acid and 2 mM EDTA) as running buffer (1).Switch UnitThe ability to reproducibly control the switch interval is critical for the separation (24). Thelimited speed at which relays can switch will not accommodate the fast switching necessary forthe PFGE separation of small DNA molecules (2-50 kb). The relays are usually controlled by acomputer. To overcome the drawbacks of electromechanical relays, high-voltage solid-stateelectronics has supplanted electromechanical relays in recently designed commercial PFGEsystems. These switching units are commonly based on the use of metal oxide semiconductorfield effect transistors (MOSFETs) in both switching and electrode voltage control circuits.These designs offer the advantages of improved reliability, the capability of high speedswitching (0.1 ms) and ample voltage (750 V) and current (0.5 amperes) ratings (1). Theseapparatuses have the ability to control the reorientation angles between electric fields.However, these instruments cannot provide fast enough switching for the improvement of theseparation of DNA molecules smaller than 50 kb.Computer ProgramCareful control of the switch interval is crucial in controlling the resolution in PFGE. Aversatile switching unit should have software with the same characteristics. The algorithmshould be fast enough so that switch times at least as short as 1 ms can be achieved and switchinterval increments should have at least 1 ms resolution. Linear switch interval ramping hasbeen the most commonly used procedure because of its simple implementation. The maximumrun time should be about two weeks to allow for the separation of very large DNA molecules.This is controlled by a computer program (1).CoolerBuffer recirculation is an important factor, as it eliminates temperature variations within thegel so as to alleviate buffer breakdown due to electrolysis. DNA molecule migration is sensitiveto temperature, and thus a uniform temperature across the gel is needed to ensure evenmigration in each of the lanes. Buffer is recirculated through the gel chamber by a reciprocatingsolenoid pump at a rate of about 450 ml/min. The buffer is chilled in its reservoir tank by coldwater (5C) circulated through a glass tubing heat exchanger. Buffer temperature is thusmaintained at 13-15C throughout a typical run (17, 24).Pulsed-Field Gel Electrophoresis (PFGE) Technique and its use in Molecular Biology410Running Conditions for PFGEPFGE separations are sensitive to a variety of different molecular and environmentalvariables. The principle significant variables are the molecular properties of the DNA, the pulsetime, the electrical field shape, the electrical field strength, the gel composition, the sampleconcentration and the temperature.1. Pulse Time: In PFGE, DNA is subjected alternately to two electrical fields at differentangles for a time called the pulse time. The molecules must presumably change direction priorto net translational motion. Each time the field is switched, larger molecules take longer tochange direction and have less time to move during each pulse, so they migrate slower thansmaller molecules. Molecules so small that their reorientation time is short compared to thepulse time will spend most of the pulse duration in conventional electrophoretic motion wheresize resolution is quite limited. As a result of this, resolution in PFGE is likely to be optimal formolecules with reorientation times comparable to the pulse time. At applied field strengths ofabout 10 V/cm, 0.1 s pulse times resolve DNA optimally in the 5-kb size range, while pulsetimes of 1,000 s at 3 V/cm are used to resolve 3-7 Mb molecules. Pulse times are selected sothat DNA molecules of a targeted size spend most of the duration of the pulse reorientingrather than moving through the gel, which accounts for the long periods of time, usually daysor weeks, needed to fractionate large DNA molecules. The chromosomal DNA molecules ofSaccharomyces cerevisiae in the 10 Mb range requires langer electrophoresis of approximatelyone week (25).2. Electrical Field Shape: A number of different electrical field configurations wereemployed in early PFGE experiments. It was apparent that certain aspects at the field shapewere critical in achieving high-resolution PFGE separations. Electrical field strength can beadjusted to tune the size range of effective PFGE resolution. The resolution of PFGE is affectedby the number and configuration of the electrodes used, because these alter the shape of theapplied electrical fields. The most critical variable appears to be the angle between the alternateelectrical fields. The most effective electrode configurations yield angles of more than 110. Acontinually increasing angle between the fields produces band sharpening that greatly enhancesthe resolution. The angle between the alternate fields is always greater than 90 where goodresolution is observed. In cases of excellent resolution, field angles typically range from 120to 150. In contrast, where poor resolution was seen, the field angles ranged typically from110 to 150 (25). Angles of 90 or smaller are not effective, probably because the DNAmolecules easily become oriented midway between the two applied fields. Angles larger than90 are more effective (25). While more complete studies on optimum angles are needed, it isclear that angles in the range of 120-150 provide very high resolution (25). Field strengthsthat decrease, or angles that increase, progressively along the direction of the net DNA motionproduce band sharpening because the molecules at the front of each DNA zone always migratemore slowly than those at the rear.411E. (HACIOLU) BASIM, H. BASIM3. Electrical Field Strength: Electrophoretic mobility is defined as the velocity per unitfield. In most ordinary electrophoresis, the mobility is independent of field strength. Thisindependence is expected if the properties of the molecules are not directly altered by aseparation process. The field strength affects mobility in two ways. The mobilities of 100-500kb DNA show an approximately linear dependence on field strength. The field strength affectsthe DNA size of the transition between the two zones of resolution (25).4. Reorientation Angle: The widening of the reorientation angle should yield sharper bandsand better resolution. The separation of yeast chromosomes is nearly identical for thosechromosomes separated with reorientation angles of 110 and 165. However, whenreorientation angles from 105 to 165 are used to separate molecules in the size range of S.cerevisiae (200-3,000 kb), there is a 4-fold difference among the DNA velocities observed withthese different angles (1). The increase in mobility obtained with smaller reorientation anglesis even more pronounced when separating larger molecules. Most commercially availablepulsed-field gel boxes use a fixed angle of 120 between the alternating fields.5. Voltage: As with switch time, the choice of the voltage used in PFGE must also be variedwith the size of the DNA to be separated. While voltage gradients of 6-10 V/cm can be used toseparate molecules up to 1 Mb, resolving molecules larger than this in pulsed field gels requiresa reduction in voltage gradient (14, 24). Separation of chromosomes from the yeast S. pombe(3 Mb, 5 Mb and 6 Mb) requires that voltage gradients do not exceed 2 V/cm. The even largerchromosomes of N. crassa (larger than 12 Mb) were separated at 1.5 V/cm (15). The practicaleffect is to increase the run times for larger DNA molecules. Thus, electrophoretic separationof N. crassa chromosomes required up to 7 days. When the voltage gradient is reduced toseparate large DNAs, switching intervals must be lengthened (15).6. Temperature: In conventional gel electrophoresis, DNA molecules were run at roomtemperature but, in PFGE, DNA was run at a low temperature (between 4C and 15C).Temperature has a dramatic effect on DNA mobility in PFGE. Temperatures between 14C and22C are generally regarded as the best compromise between speed and resolution while gelscan be run at room temperature, it is usually necessary to circulate the buffer through a heatexchanger to dissipate the heat generated by the voltage gradients used during most pulsedfield runs (2, 25). The velocity of lambda DNA at 34C is twice that at 4C. However, gels runat temperatures as high as 34C show diminished resolution (1).7. Switch interval: The single most important determinant of mobility in PFGE is theinterval at which the direction of the electric field is switched. If the switching interval isincreased beyond the time required for a fragment to reorient, then the fragment will spend alarge portion of the gel run migrating, as in conventional electrophoresis, with a resulting lossin resolution. The choice of an appropriate switching interval for PFGE must reflect the sizerange of the fragments to be resolved. Birren et al. (24) have measured the velocity of DNAmolecules from 50 to 1,000 kb in PFGE with switch times of from 5 to 300 s. The highestPulsed-Field Gel Electrophoresis (PFGE) Technique and its use in Molecular Biology412resolution for molecules of a given size is obtained by using the shortest switch intervals whichpermit separation of the complete size range of the fragments (26).8. Agarose Concentration: The agarose concentration will affect the separation obtainedwith PFGE. Faster DNA migration occurs in gels of lower agarose concentration. The DNAmonomer (48.5 kb) migrates 50% faster in PFGE of 0.6% agarose compared to a 1% gel. TheDNA bands which are quite diffuse in the 0.7% gel become increasingly sharp as the agaroseconcentration is raised in 1.4% and 1.8% gels over identical times. The distance migrated bythe identical samples demonstrated the decrease in velocity as the agarose concentration isincreased (24).9. Restriction Enzymes: The ability of restriction enzymes (REs) to cut DNA at a specificsequence of bases has greatly stimulated the growth of recombinant DNA technology. Over1,900 REs are known, and of there 275 are available from companies based around the world(27). The common restriction enzymes, EcoR I and Hind III, digest bacterial and mammalianDNA to fragments averaging approximately 4 kb in sizes much too small for PFGE. For thisreason, it is advisable to use enzymes which have relatively few sites and give larger fragmentsfrom the target DNA in PFGE (27). Any enzyme producing a large number of small fragments(smaller than 10 kb) is unlikely to be useful for PFGE and mapping. A major factor in selectingsuitable restriction enzymes is the base composition (%G+C content) of the target DNA.Analysis by PFGE and rare-restriction enzymes have been useful for obtaining relevantinformation on genome size, characterizing various strains at the DNA level (28), following thegenetic history of a particular strain, constructing physical and genetic maps of bacterialchromosomes and studying chromosomal dynamics among bacteria (29-31). Enzymes thatrecognize sequences larger than 6 bp are potentially useful in genome mapping because theygenerate large fragments (27). At present, there are nine restriction enzymes commerciallyavailable with an 8-bp recognition sequence. Of these, Not I, Sfi I, Srf I, Ase I, Pac I and Swa Iare rare-cutters in genomes with a G+C content of about 35-55%. Below and above thesemargins the number of fragments becomes too small and too large, respectively. Pac I(AATTAATT), Swa I (ATTTAAAT), Pme I (GTTTAAAC) and Sse 83871 (CCTGCAGG) are new onthe market (32). On the other hand, Pac I and Swa I enzymes should be useful especially forgenomes with a G+C contents in the range of 45-65% (32). The 4-bp pair sequence 5-CTAG-3 seems to be selected against in most bacterial genomes (33). This tetranucleotide is part ofthe recognition sequence of Spe I (A/CTAGT), Xba I (T/CTAGA), Nhe I (G/CTAGC) and Avr II(C/CTAGG). CTAG is found infrequently in most prokaryotes and restriction endonucleases thatinclude this sequence in their recognition sequence cut bacterial genomes infrequently (32).PFGE of large fragments of DNA generated using infrequently cutting Swa I and Pac Irestriction endonucleases were used in genome analysis of Xanthomonas axonopodis pv.vesicatoria, a causal agent of leaf spot disease of pepper and tomato, and optimal conditionsfor digestion in the genome analysis were determined (26, 34-36).413E. (HACIOLU) BASIM, H. BASIMPulsed-Field ApplicationsFull understanding of a biological system requires knowledge of the structure and functionof the genes and their arrangement on the chromosome. PFGE has been used to separate DNAmolecules as large as 12 Mb. The ability to analyze such large fragments will greatly facilitatethe construction of physical maps (1).The ability to separate, isolate and analyze megabase size fragments of DNA is alreadyproviding insights into the genome organization of organisms as diverse as bacteria and humans(2).PFGE is used in the following areas:1. The advent of PFGE techniques for the resolution of large DNA molecules has provideda new analysis approach for bacterial genomes (37). The PFGE of DNA fragments obtainedusing different enzymes is a powerful technique for quick resolution of the bacterial genomeinto a small number of large fragments.2. The PFGE of DNA fragments obtained by using endonucleases produce a discrete patternof bands useful for the fingerprinting and physical mapping of the chromosome (38).3. The PFGE technique is useful to establish the degree of relatedness among differentstrains of the same species (38).4. PFGE has proved to be an efficient method for genome size estimation and theconstruction of chromosomal maps, as well as being useful for the characterization of bacterialspecies (39-41). PFGE technology has proven invaluable for the accurate estimation of genomesize and in the construction of physical maps of a diverse range of prokaryotic organisms(42,43).5. PFGE is a powerful tool for genome characterization and has led to the construction ofthe physical map of more than 180 bacterial chromosomes (44).6. PFGE has proven extremely powerful in the analysis of large DNA molecules from avariety of sources including intact chromosomal DNAs from fungi (16), parasitic protozoa (45)and specifically fragmented genomes of bacteria (26, 38) and mammals (5, 6).7. PFGE simplifies many previously laborious investigations and makes possible many newinvestigations. This technique has been used extensively in application to all organisms frombacteria to viruses (2).8. Yeast Artificial Chromosome (YAC) libraries have been constructed by PFGE (2).9. PFGE experiments are used in the construction of transgenic mice (2).10. PFGE has also shown itself useful in the study of radiation-induced DNA damage andrepair, size organization and variation in mammalian centromers (2, 46).Pulsed-Field Gel Electrophoresis (PFGE) Technique and its use in Molecular Biology41411. Many investigations directly involve studies of genome organization. For example, indetermining the physical proximity of two genes, while determining the size of very large genesor when identifying the location of chromosomal breaks (2, 46).12. PFGE will play a major role in the mapping of the human genome (47). The physicalmapping of a human chromosome involves ordering and measuring the distances between a setof DNA markers that are unique to that chromosome. Given the large sizes of the chromosomesinvolved (50,000-300,000 kb), PFGE is the method to pursue (47).13. PFGE is used to determine the order of markers more precisely than is possible withgenetic linkage analysis, and with a PFGE map available, new markers can be quickly localizedwithin that region (32).14. A new mutation can be mapped by cloning the gene, followed by restriction analysis andhybridization, to a set of ordered restriction fragments by PFGE (47).15. PFGE will greatly facilitate the precise selection of large DNA fragments for cloning. REswhich are specific for cutting infrequently occurring sequences, are used to create large DNAfragments which are then separated by PFGE. By blotting and hybridization the fragmentscontaining the desired gene are determined. This region is recovered from the gel and cloned(2, 23).16. PFGE allows for easy isolation of the individual restriction fragments for furtherrestriction mapping, gene insertion and functional gene mapping (13).ResultsThe developments in PFGE over the past decade have led to an ingenious collection ofworkable designs. Todays instruments resolve DNA several orders of magnitude larger thanwas possible by conventional electrophoresis, permitting the direct study of intactchromosomes from yeasts and human parasites. Investigations now center on a betterunderstanding of these techniques, which will support the design of new generations of devicesto separate even larger fragments and, eventually, entire human genomes.The analysis of entire genomes represents a revolutionary approach to genetics, and theavailability of vast amounts of information from these projects will allow new conceptualapproaches in many areas of biological research. It is clear that at the present time, no singletechnique or strategy is sufficient for preparing a map of an intact human chromosome.However, the combined use of many powerful new techniques brings the analysis ofmammalian genomes within reach.PFGE has made possible the development of the YAC cloning system and will play a majorrole in the mapping of the human genome. The PFGE technique will be useful for establishingthe degree of relatedness among different strains of the same species.415E. (HACIOLU) BASIM, H. BASIMFuture applications for PFGE techniques may include protein separations and nucleic acidsequencing and studies of DNA topology. PFGE allows physical-map construction for virtuallyany organisms. PFGE techniques should soon provide physical maps and their applications fora wide number of microorganism. As physical mapping methods are rapidly displacing geneticmethods for chromosome assignment, it is essential to understand the behavior of circular DNAspecies on pulsed-field gels.Analysis with PFGE can be used to establish long-range maps based on anonymous markersthat have been shown to be genetically linked. Ideally, the targeted region is saturated withmarkers, allowing the construction of several overlapping maps. However, for plant genomes,markers are rarely clustered with sufficient density to allow the immediate construction ofdetailed maps based on several markers.References1. Lai, E., Birren, B. W., Clark, S. M., Simon, M. I., Hood, L. Pulsed-Field Gel Electrophoresis. 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Jr., Freifelder, D. Microbial Genetics. Jones and Bartlett Publishers, Second Edition,45-47, 1994.9. Kaufmann, M. E., Pitt, T. L. Methods in Practical Laboratory Bacteriology. Edited by H. Chart, FL, CRC Press Inc.,83, 1994.10. Levene, S. D. Methods in Molecular Biology, Pulsed-Field Gel Electrophoresis. Eds. M. Burmeister and L.Ulanovsky, Totowa, NJ, The Humana Pre