BT-403 MOD-III NKJ LECTURE : ISOLATION OF DNA Whenever the quantity of DNA that you need to work with

can not be obtained directly or through PCR, or you need a constant source of identical DNA, cloning of the DNA is the solution. SOURCES OF DNA There are four primary sources of DNA: Cells, Organelles, Plasmids, and Viruses. There are separate considerations for the isolation of DNA from each of these. Each of the DNA sources must be purified away from proteins, RNA, carbohydrates and lipids. Sometimes DNAs must be purified away from each other. For example, in isolating organellar DNA it is easier to first isolate the organelle separate from the nuclei and then extract the organellar DNA. In other cases, such as in plasmid isolstion from bacteria, the chromosomal DNA and plasmid DNA reside in the same cell and must be separated by physical differences between the two. CONCENTRATING CELLS Centrifugation is a method that allow cells to be concentrated and separated from the media in which they are grown. Many media are complex and so this provides a good first step in removing potential contaminants.

DNA PURIFICATION FROM CELLS

Once cells are concentrated and separated from growth media, the cells can be lysed. There are various way to lyse cells. Source Bacteria Lysis Methods Lysozyme - degrades polysaccharide cell walls SDS - detergent for cell membranes Freeze/Thaw several times French Press Osmotic Sonication Detergent Osmotic Sonication Detergent Several enzymes to break down cell walls Mechanical grinding

Organelles

Animal Cells Plant Cells

REMOVING CONTAMINANTS

Lysis and centrifugation to remove cell debris.

Contaminant Carbohydrates

Method of Removal CTAB + NaCl

Cell Walls & Cellular Debris Centrifugation

Protein

Phenol/CHCl3 extraction Protease Guanidinium Thiocyanate & Silica CsCl gradient with ethidium bromide RNase CsCl gradient with ethidium bromide

RNA

CTAB extraction

Phenol extraction

Glass milk purification of DNA using ITC.

CONCENTRATING DNA AND CHANGING SOLVENTS Ethanol precipitation (70% + salt to neutalize negative charges) Also precipitates RNA and some protein.

Precipitation of DNA with ethanol.

Reduce aqueous volume by extracting with butanol. Butanol excludes DNA and absorbs water.

PLASMID ISOLATION Plasmid DNA must be isolated free from chromosomal DNA. There have been a number of methods described over the years but the most popular and commonly used method is the Alkaline Lysis method. 1- Pellet cells by centrifugation, discard supernatant. 2- Resuspend in Solution I (cloudy suspension, no clumps) Glucose - osmoticant Tris - buffer EDTA - chelated divalent metal cations (inhibits RNases and DNases) Lysozyme - optional, breaks down cell wall polysaccharide 3- Mix gently with Solution II (clear, viscous) SDS - Detergent to lyse cell walls NaOH - raises pH to over 11.3 => denatures DNA 4- Mix gently with Solution III (white clumps, like small curd cottage cheese) 3M KAc - lowers pH, neutralizes DNA which renatures quickly in the high salt. Causes the complementary single strands to zip together quickly while the genomic DNA form an insoluble glob because it can't find the right complementary strand.

5- Centrifuge to pellet the cellular debris and the insoluble genomic DNA mass.

6- Ethanol precipitate to change the solvent. 7- Resuspend in a buffer. (still contains some RNA and Protein as contaminants)

FURTHER PURIFICATION Various column types, Qiagen, Promega, ...

CsCl gradient with ethidium bromide

HOW TO DETERMINE THE QUANTITY AND QUALITY OF YOUR DNA The quality of DNA can be determined from a wavelength scan between 260 and 320 nm. DNA and proteins do not absorb at 320 nm and any significant readings usually come from the light scatter of insoluble particles, such as denatured proteins. Proteins absorb at a maximum around 280 nm, while nucleic acids, such as DNA and RNA, show maximal absorption at 260 nm. The quality of DNA is often initially estimated by looking at the ratio of the absorbance at 260 nm / 280 nm. A260 / A280 Ratio < 1.8 1.8 < Ratio < 1.9 Ratio > 2 Quality Contains protein contamination no contamination DNA may contain RNA

Absorbance peak or shoulder at 270 nm Phenol contamination The quantity of pure nucleic acids can be estimated from the following Nucleic Acid Form Double -stranded DNA Single-stranded DNA RNA NA Concentration at 1 A260 50 g/ml 37 g/ml 40 g/ml