(en)lecture 6 - nucleotide structure part...

國立交通大學生物科技學系蘭宜錚老師 1

Lecture 6• Structure of nucleic acids part 2

DBT2117: Biochemistry (I)

1

Meselson & Stahl experiment shows DNA replication to be semiconservative

• Through Avery (1944) & Hershey/Chase (1952), DNA is shown to be the reproductive biomolecule

• Watson & Crick proposed the double helix model in 1953. In their model, they proposed not only the structure of DNA, but more importantly how DNA is copied and reproduced through a templated process.

• 1958 – Meselson & Stahl provided the experimental test to show the semi‐conservative nature of DNA replication.

• This was achieved through observation of DNA density as the result ofusing different Nitrogen isotopes.

• Density difference was observed by CsCl density gradientcentrifugation. (using ultracentrifuge to spin Cs+Cl‐ solution)

• Since Cs+ is heavy (atomic mass 133), it is pushed to the bottom of the tube. An equilibrium gradient (of Cs+ concentration and therefore density) is formed. Top of the tube being mostly water, and bottom of the tube contains more Cs+ ions.

• Molecules with higher density than water moves down until the density of such molecule equals the density of the Cs+ gradient

http://pirate.shu.edu/~rawncarr/Enzyme2/Enzy2.htm

http://umaine.edu/connelllab/category/antarctic‐fungi‐blog/page/2/


Differential centrifugation

• Separation & purification is a key technique is all areas of chemistry and biology.• Centrifugation provides an easy method for separating some biological parts.

• Differential centrifugation applies different centrifugation speed to pellet (or sediment)different components of the cell.

http://www.slideshare.net/raiuniversity/bsc‐biotech‐ii‐bat‐unit‐2‐centrifugation

Basis of separation:• Size• Shape• Density

Separation of biological components

Samples are first homogenized (breaking cell membrane)Then, these homogenates are fractionated using differential centrifugation. More detailed separation can be achieved using density gradients.


Meselson & Stahl experiment shows DNA replication to be semiconservative

DNA is double stranded at pH7single stranded at pH12

Cells grown using light Nitrogen (14N) or heavy Nitrogen (15N) only have one type of DNA.

DNA formed using 15N is higher in density.

Cells grown using 15N was switched to 14N. As expected, a hybrid DNA appeared. This excluded conservative model

When DNA was separated into single stranded. Only two types of DNA were observed. Heavy and Light (increasing light with exactly double the amount each generation). At 2nd generation, ¾ were light, ¼ was heavy. This result excluded the possibility of dispersive.

6 bonds can rotate (#4 is limited in its available rotation). Structural variation in DNA reflect 3 things: • Rotation of sugar phosphate bonds (bond 1, 2, 3, 5, 6)• Free roation of bond 7: syn or anti

• different possible conformations of the deoxyribose: endo or exo.

Nucleic acids have many bonds that can rotate


DNA has different forms A, B, and Z

The form that Watson & Crick studied was the B form DNA.

This is because B form DNA is the predominant form in aqueous environment. A form is observed in low humidity.

A & B forms represent the major forms of polynucleotide secondary structure.

Most polynucleotides are in A or B forms

Most DNA (double stranded) exist in B form, with 10.5 bases per turn having been observed.

B form DNA is most stable in aqueous environment because H2O fills in the minor groove. This hydrogen bonding stabilizes B form.

Double RNA & RNA‐DNA hybrids exist in A form. B form is NOT favored for RNA because the 2’‐OH would get too close to the phosphate.

Most polynucleotides are not straight helixes. Rather, they are tilted.

Local sequences also have an effect on the local structure of the DNA. Therefore, the A & B forms should only be thought as an “average” value.


Z form

Z‐DNA is mostly found in polynucleotides with alternating purines and pyrimidines in each strand.

In Z‐form, pyrimidines are always anti and purines are always syn.

More notably, Z‐form has a left‐handed helical structure.

The exact function of this form is still not certain.

5’‐CGCGCG‐3’

Single stranded nucleic acids

Conformations of single‐stranded nucleic acids.

(a) The random coil structure of denatured single strands. There is flexibility of rotation of residues and no specific structure.

(b) Stacked‐base structure adapted by non–self‐complementary single strands under “native” conditions. Bases stack to pull the chain into a helix, but there is no H‐bonding.

(c) Hairpin structures formed by self‐complementary sequences; the chain folds back on itself to make a stem–loop structure.


• Self‐complementarity in a base sequenceallows a chain to fold back on itself and form abase‐paired, antiparallel helix or hairpinstructure

• Double hairpins, often called cruciform (cross‐like) structures, can be formed.

• To form this structure, the sequence mustbe palindromic.

• The word palindrome is of literary originand usually refers to a statement thatreads the same backward and forward,such as “Able was I ere I saw Elba.”

Hairpins and cruciforms

FIGURE 4.29A palindromic DNA sequence. A palindrome is strandwise symmetrical about a center of symmetry. Note that in the portion in brown and blue, both blue segments read the same 5 3 , as do both brown segments. The sequence is shown in its extended and cruciform conformations. Two bases that will pair with each other in the cruciform conformation are given the same number.

Hairpins and cruciforms

Palindromic sequence


4 - 13

• RNA molecules are usually single‐stranded.

• Most have self‐complementary regionsthat form hairpin structures.

• Some have well‐defined tertiarystructures.• These 3D structures have importance

to their specific functions and roles.

Most RNA are usually single stranded

http://www.nature.com/scitable/blog/bio2.0/dna_origami

Using properties of DNA, we can design them into shapes


Relative lengths and functions of RNA

Relative lengths and functions of RNA


Supercoiling for circular DNA

• Typical bacteria and archaea have a circular chromosome.

• Plasmids are also circular DNA (shorter than chromosome)

• These circular DNA are typically supercoiled in vivo.

Relaxed

Supercoiled

Most naturally occurring circular DNA have left‐handed superhelical twists. This is called negative supercoiling.

Supercoiling for circular DNA


Denaturation of double stranded DNA to single stranded

Denaturation of DNA.

a)When native (double‐stranded) DNA is heated above its “melting” temperature, it isdenatured (separates into single strands). The two randomcoil strands have a higher entropy than the double helix.

b)At low T, ΔG is positive and denaturation of DNA is not favored. As T increases,‐T ∆ S overcomes ∆ H, making ∆ G negative and denaturation favorable.

The midpoint of the curve marks the “melting” temperature, Tm, of DNA.

Denaturation of DNA.

c)Absorption spectra of native and denatured DNA show that native DNA absorbs lesslight than denatured DNA, with the maximum difference occurring at a wavelength of 260 nm. This hypochromicity of doublestranded DNA can be used to distinguish between native and denatured forms.

d)The change in absorbance can be used to follow the denaturation of DNA astemperature increases. An abrupt increase in absorbance, corresponding to the sudden “melting” of DNA, is seen at Tm.

Denaturation of double stranded DNA to single stranded


Effects of GC% on melting temperature

Effect of base‐pair composition on the denaturation temperature of DNA.

The graph shows the rise in “melting” temperature of DNA as its percent (G + C)increases.

More GC a DNA molecule has, the more energy is required to separate the double strands. This is because GC have more H‐bonding

DNA manipulations and molecular biology

Recombinant DNA techniques are used to study nucleic acids and their transcription and translation into proteins

Some Recombinant DNA techniques include: 1. Gene cloning2. Chemical synthesis of oligonucleotides for use as primers3. DNA sequence analysis4. Site‐directed mutagenesis5. Polymerase chain reaction (PCR)6. Homologous recombination

Some applications for recombinant DNA techniques are:1. Recombinant protein production (such as insulin, growth hormone, vaccines, etc)2. Genetic modification of crops (insect and herbicide resistant, cold & draught resistant)3. Synthetic biological functions (biofuel & biochemicals, biosensing, etc)

4. To study specific functions of genes and/or proteins (purification of human enzymeusing E.coli for study)

5. Many more applications involved in biotechnology, agriculture, medicine, research.


Creating recombinant DNA

DNA can be manipulated through amplification and then “cut and paste”

PCR (polymerase chain reaction) is used to amplify DNA.

In order to manipulate DNA, you need sufficient concentration of it.

PCR amplifies a target region by using primerswhich sets the boundary of the amplification

Creating recombinant DNA

DNA can be manipulated through amplification and then “cut and paste”


Oligonucleotide synthesis – a chemical synthesis

• Primers are important for various molecular biology & biochemistry experiments.

• Primers are made using chemical synthesis from 3’‐5’ direction

• This process is now automated

• Currently, each base is about $0.3/base (9 NTD)

• Primers are typically less than 60 bps

Sanger Sequencing ‐ Dideoxynucleotide

Fred Sanger developed a sequencing method using ddNTP (Dideoxynucleotide triphosphate)

ddNTP terminates DNA polymerization because it does not have a 3’‐OH group to form the next phosphate dister bond.

Individually add ddATP, ddTTP, ddCTP, or ddGTPinto a polymerization reaction (PCR). Note that PCR still contains the dNTP necessary for polymerization.

To sequence a DNA, 4 PCR are conducted.

Every time when an A is supposed to be added to the chain, there is a chance that ddATP is used instead of dATP. If ddATP is incorporated, then that chain is terminated.

Same idea for T, C, and G.


Sanger Sequencing ‐ Dideoxynucleotide

Sanger sequencing has been improved by adding a different fluorescent tags on each ddNTP. Each type of ddNTP emits a different color light.

Step 1: PCR reaction: contains template, primer, polyermase, dNTP, colored ddNTP Step 2: Separation of DNA products by

capillary gel

Step 3: Detection with Laser & detector for observation of ddNTPcolor.

Using Sanger Sequencing

Currently, Sanger sequencing can generate about 700 bases reads per reaction.

This can be used for quick sequencing of small fragments

For genome scale sequencing, next generation sequencing techniques are used instead. (because they are significantly faster due to ability of running in parallel)

(en)lecture 6 - nucleotide structure part...

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