ISOLATION OF CHROMOSOMAL DNA FROM BACTERIA

Introduction:

The bacterial cell does not have its genetic material enclosed in a membrane bounded nucleus

and hence is a prokaryote. Instead, they contain highly compact structure called nucleoid.The

chromosome of  E. coli and presumably of most bacteria is a single super coiled double-stranded

circular DNA molecule complexed with a specific amount of protein and variable amount of 

RNA.The  E.coli chromosome is well studied. For  E. coli the total length of the circular 

chromosomal DNA is about 1300 µm where as the rod-shaped bacterium has a diameter and

length of about 1 and 3 µm, respectively. Therefore the bacterial chromosomal DNA must be

highly folded when it is in a cell. The genome content is about 5000 kbp consisting of 2800

genes. Two important features of the structure of  E. coli chromosome have been revealed by

electron micrograph studies, a) The DNA is arranged in a series of loops and b) each loop is

super coiled.

The physical organization of bacteria (prokaryotes) is simple as compared to eukaryotes. In

 bacteria, there are two distinct classes of cell envelopes known as Gram-positive and Gram-

negative. Gram-positive cell envelopes consist of plasma membrane and peptidoglycan layer 

(cell wall) where as the architecture of Gram-negative cell envelope is more complex consisting

of plasma membrane, periplasmic space, peptidoglycan and outer membrane. E.coli is an

example of Gram-negative bacterium. Therefore its cell wall is more complicated than that of 

Gram-positive cells. In order to isolate chromosomal DNA from  E. coli, the cell envelope

structure needs to be weakened and ruptured. Treatment of cell with various reagents such as

trypsin (which hydrolyze proteins), detergents (which remove lipids), and lysozyme

(muramidase) causes dissolution of peptidoglycan layer. The isolation of  E.coli chromosomal

DNA is based on the following principle:

 

Principle:

The cells are first digested with lysozyme in order to weaken the cell walls then ruptured with

the detergent sodium doddery sulphate. In the extraction medium high salt concentration (0.15

mol/litre helix NaCl) is used. This helps to prevent strand separation of the double of the

DNA.EDTA is also present and his chelate metal ions (such as Mg++, Ca++) needed for DNase

and so inhibits the activity of the enzyme. Protein is denatured by the treatment with the buffer-

saturated phenol. Extraction is carried out with the organic solvent mixture (phenol: chloroform:

isoamyl alcohol) to remove denatured protein contaminants isoamyl alcohol is present as an

antifoaming agent. After removal of the protein, DNA is removed by the precipitation with

ethanol or isopropanol.Further incubation of the chromosomal DNA solution with RNase (this

should be DNase free by heat activation) should ensure that contamination of the DNA by RNA

is kept at minimum level. The conc. Of the isolated DNA may be determined by UV absorption

spectrum at 260 nm and 280 nm can be used to detect protein contamination. The quality of the

chromosomal DNA may be further assessed through digestion by restriction enzymes followed

 by agarose gel electrophoresis.

Requirements:

(Media and solutions should be sterile. Sterilization by autoclaving needs to be done at 15 lbs

for 15 minutes)

 E. coli DH5α strain,LB medium and LB plates, Saline –EDTA (0.15M NaCl,0.1M EDTA

adjusted to pH 8.0), Lysozyme soln.(10 mg/ml),1.0 M Tris.HCl (pH 8.0),10% sodium dodecyl

sulphate (SDS),50 mM Tris buffer (pH 8.0),saturated phenol,Chloroform,isoamyl alcohol,TE

 buffer (10 mM Tris HCl pH 8.0,1 mM EDTA pH 8.0),Iso propanol, Dehydrated ethanol ,DNase

free RNase solution(10 mg/ml),microfuge tubes, micro tips, UV spectrophotometer and quartz

cuvettes

 

Procedure:

The following protocol may be used for small-scale preparation of  E. coli chromosomal DNA:

Pick a single colony of  E. coli DH5α strain from a freshly grown plate and transfer it into 20 ml

of LB broth in a 250 ml of flask. Incubate the culture for 16-20 hours at 37˚C with vigorous

shaking (200-250 cycles/minute in a rotary shaker).(More than one group may be involved in

the same isolation procedure with this culture).

Harvest cells from the above 1.5-2.0 ml stationary phase culture in sterile microfuge tube.

Decant the media from the cell pellets, stand the tubes in an inverted position for one minute to

allow the last traces of media to drain away(The cell pellet may be washed here using 10mM

Tris HCl pH 8.0)

Resuspend each cell pellet in approximately 0.8ml saline EDTA buffer thoroughly. Add 50 µl

freshly prepared lysozyme soln.mix well. Incubate at 37˚C for 20 minutes.

Add 0.2 ml 10% SDS, mix well by inversion, and incubate in water bath at 60˚C for 15 minutes.

Extract once with buffer-saturated phenol.

Extract once with phenol: chloroform: isoamyl alcohol (25:24:1)

Transfer the upper aqueous phase to a sterile microfuge tube. Precipitate DNA by adding equal

volume of isopropanol.

Wind out the DNA fibers on the glass rod. Squeeze as much liquid as possible from the spooled

mass by pressing it against the side of microfuge tube.

Dissolve the DNA fibers in 500 µl TE buffer 

Add DNase free RNase solution to a final conc. of 40 µg/ml and incubate at 37˚C for 30 minutes,

with occasional shaking.

Extract once with equal volume of phenol: chloroform: isoamyl alcohol (25:24:1)

Precipitate DNA from the upper aqueous layer with 2 volume of ethanol. Spool out the DNA and

redissolve it in 50 µl TE (pH 8.0).Store at 4 ˚C for further use.

 

Calculate the conc. of DNA using UV spectrophotometer (50 µg/ml of DNA) has an extinction

of 1.0 at 260 nm in 1 cm cuvette).Also find out the E260/E280 ratio.

References:

Oishi, M and Cosloy, SD.1972.The genetic and biochemical basis of the transformability of 

 Escherichia coli K12.Biochem.Biophys.Res.Cummun.49:1568

Worcel, A.and Burgi, E.1972.On the structure of the folded chromosome of    Escherichia

coli.J.Mol.Bio.71:127

Sinden, RR.And Pettijohn, DE.1981.Chromosomes in living  Escherichia coli cells are

segregated into domains of supercoiling.Proc.Natl.Acad.Sci. USA 78:224

Plummer, DT.1988.an introduction to Practical Biochemistry (Chapter 16), (Third Edition),Tata

McGraw-Hill, New Delhi.