GENE CLONING AND DNA ANALYSIS

基因工程與原理

Chapter 3 Purification of DNA from Living Cells

3.1 Preparation of total cell DNA3.1.1 Growing and harvesting a bacterial culture3.1.2 Preparation of a cell extract3.1.3 Purification of DNA from a cell extractRemoving contaminants by organic extraction and enzyme digestionUsing ion-exchange chromatography to purify DNA from a cell extract3.1.4 Concentration of DNA samples3.1.5 Measurement of DNA concentration3.1.6 Other methods for the preparation of total cell DNA3.2 Preparation of plasmid DNA3.2.1 Separation on the basis of size3.2.2 Separation on the basis of conformationAlkaline denaturationEthidium bromide–caesium chloride density gradient centrifugation3.2.3 Plasmid amplification3.3 Preparation of bacteriophage DNA3.3.1 Growth of cultures to obtain a high λ titer3.3.2 Preparation of non-lysogenic λ phages3.3.3 Collection of phages from an infected culture3.3.4 Purification of DNA from 2 phage particles3.3.5 Purification of M13 DNA causes few problems

Figure 3.1The basic steps in preparation of total cell DNA from a culture of bacteria.

3.1 Preparation of total cell DNA

3.1.1 Growing and harvesting a bacterial culture

Defined medium

Complex or undefined

Tryptone 蛋白胴 : digestion of casein酪蛋白 by the protease trypsin; amino acids and small peptides

Yeast extract: nitrogen, sugar, inorganic and organic nutrients

Figure 3.2Estimation of bacterial cell number by measurement of optical density (OD). (a) A sample of the culture is placed in a glass cuvette and light with a wavelength of 600 nm shone through. The amount of light that passes through the culture is measured and the OD (also called the absorbance) calculated as:

(b) The cell number corresponding to the OD reading is calculated from a calibration curve. This curve is plotted from the OD values of a series of cultures of known cell density. For E. coli, 1 OD unit = 0.8 × 109 cells/ml.

1 OD unit = log10

Intensity of transmitted light

Intensity of incident light

Figure 3.3Harvesting bacteria by centrifugation.

Figure 3.4Preparation of a cell extract. (a) Cell lysis. (b) Centrifugation of the cell extract to remove insoluble debris.

3.1.2 Preparation of a cell extract

Lysozyme: digest the cell wallEDTA: remove magnesium, inhibit DNaseSDS: disrupt the cell membrane

Figure 3.5Two approaches to DNA purification. (a) Treating the mixture with reagents which degrade the contaminants, leaving a pure solution of DNA. (b) Separating the mixture into different fractions, one of which is pure DNA.

Ion-exchange chromatography

Figure 3.6Removal of protein contaminants by phenol extraction.

or phenol and chloroform (1:1)

Protease (pronase or proteinase K) before phenol extraction

Ribonuclease degrades RNA

Figure 3.7DNA purification by ion-exchange chromatography. (a) Attachment of DNA to ion-exchange particles. (b) DNA is purifiedby column chromatography. The solutions passing through the column can be collected by gravity flow or by the spincolumn method, in which the column is placed in a low-speed centrifuge.

DNA and RNA are negatively charged

Figure 3.8Collecting DNA by ethanol precipitation. (a) Absolute ethanol is layered on top of a concentrated solution of DNA. Fibers of DNA can be withdrawn with a glass rod. (b) For less concentrated solutions ethanol is added (at a ratio of 2.5 volumes of absolute ethanol to 1 volume of DNA solution) and precipitated DNA collected by centrifugation.

Ethanol precipitation

Monovalent cation (Na+)-20 or less℃Absolute ethanol

3.1.4 Concentration of DNA samples

3.1.5 Measurement of DNA concentration

Ultraviolet (UV) absorbance spectrophotometry

1 OD (A260)= 50 mg/ ml dsDNA

A260/ A280 ~ 1.8

Figure 3.9The CTAB method for purification of plant DNA.

3.1.6 Other methods for the preparation of total cell DNA

Cetryltrimethylammonium bromide (CTAB)

Figure 3.10DNA purification by the guanidinium thiocyanate and silica method. (a) In the presence of guanidinium thiocyanate, DNA binds to silica particles. (b) DNA is purified by column chromatography.

Figure 3.11Preparation of a cleared lysate.

3.2 Preparation of plasmid DNA

3.2.1 Separation on the basis of size

Figure 3.12Two conformations of circular double-stranded DNA: (a) supercoiled—both strands are intact; (b) open-circular—one or both strands are nicked.

3.2.2 Separation on the basis of conformation

Covalently closed-circular (ccc) Relaxed

Figure 3.13Plasmid purification by the alkaline denaturation method.

Non-supercoiled DNA is denatured

Figure 3.14Caesium chloride density gradient centrifugation. (a) A CsCl density gradient produced by high speed centrifugation. (b)Separation of protein, DNA, and RNA in a density gradient.

Figure 3.15Partial unwinding of the DNA double helix by EtBr intercalation between adjacent base pairs. The normal DNA molecule shown on the left is partially unwound by taking up four EtBr molecules, resulting in the “stretched伸長” structure on the right.

Figure 3.16Purification of plasmid DNA by EtBr–CsCl density gradient centrifugation.

Figure 3.17Plasmid amplification.

Some multicopy plasmids can replicate in the absence of protein synthesis

Figure 3.18Preparation of a phage suspension from an infected culture of bacteria.

3.3 Preparation of bacteriophage DNA

The maxinum titer for λ is 1010 /ml; ~ 500 ng DNA

Figure 3.19Induction of a λ cIts lysogen by transferring from 30°C to 42°C.

3.3.1 Growth of cultures to obtain a high λ titer

Temperature-sensitive (ts)

Figure 3.20Achieving the right balance between culture age and inoculum size when preparing a sample of a non-lysogenic phage.

3.3.2 Preparation of non-lysogenic λ phages

Deletions of the cI and other genes.

Figure 3.21Collection of phage particles by polyethylene glycol (PEG) precipitation.

3.3.3 Collection of phages from an infected culture

Figure 3.22Purification of λ phage particles by CsCl density gradient centrifugation.

3.3.4 Purification of DNA from λ phage particles

Figure 3.23Preparation of single-stranded M13 DNA from an infected culture of bacteria.

3.3.5 Purification of M13 DNA causes few problems

1012 M13/ml