1.Maximize DNA recovery
3.Remove or inhibit nucleases
4.Maximize the quality of DNA
5.Double strand vs. Single strand (RFLP or PCR)
- The RFLP procedure on requires a minimum of 50 ng of high molecular weight double stranded DNA. This is the equivalent of approximately 2 ul of blood. The number of intact sperm ( 3 pg/sperm) is approximately 20,000.
- The PCR reactions call for on average 1 ng of DNA (single or double stranded).
- This is the equivalent of 1/20 of 1 ul of blood, or 350 sperm.
- Many of the commercially available kits are sensitive below 1 ng of DNA (100-250 pg).
- Organic (Phenol-Chloroform) Extraction
- Non-Organic (Proteinase K and Salting out)
- Chelex (Ion Exchange Resin) Extraction
- FTAÔ Paper (Collection, Storage, and Isolation
Perhaps the most basic of all procedures in forensic molecular biology is the purification of DNA. The key step, the removal of proteins, can often be carried out simply by extracting aqueous solutions of nucleic acids with phenol and/or chloroform.
- Cell Lysis Buffer – lyse cell membrane, nuclei are intact, pellet nuclei.
- Resuspend nuclei, add Sodium Dodecly Sulfate (SDS), Proteinase K. Lyse nuclear membrane and digest protein.
- DNA released into solution is extracted with phenol-chloroform to remove proteinaceous material.
- DNA is precipitated from the aqueous layer by the additional of ice cold 95% ethanol and salt
- Precipitated DNA is washed with 70% ethanol, dried under vacuum and resuspended in TE buffer.
- Cell Lysis Buffer – Non-ionic detergent, Salt, Buffer, EDTA designed to lyse outer cell membrane of blood and epithelial cells, but will not break down nuclear membrane.
- EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating agent of divalent cations such as Mg2+. Mg2+is a cofactor for Dnase nucleases. If the Mg2+is bound up by EDTA, nucleases are inactivated.
- Proteinase K – it is usual to remove most of the protein by digesting with proteolytic enzymes such as Pronase or proteinase K, which are active against a broad spectrum of native proteins, before extracting with organic solvents. Protienase K is approximately 10 fold more active on denatured protein. Proteins can be denatured by SDS or by heat.
- Phenol/Chlorform – The standard way to remove proteins from nucleic acids solutions is to extract once with phenol, once with a 1:1 mixture of phenol and chloroform, and once with chloroform. This procedure takes advantage of the fact that deproteinization is more efficient when two different organic solvents are used instead of one.
- Also, the final extraction with chloroform removes any lingering traces of phenol from the nucleic acid preparation.
- Phenol is highly corrosive and can cause severe burns.
- Phenol – often means phenol equilibrated with buffer (such as TE) and containing 0.1% hydroxyquinoline and 0.2% b-mercaptoethanol (added as antioxidants. The hydoxquinoline also gives the phenol a yellow color,making it easier to identify the phases (layers).
- Chloroform – often means a 24:1 (v/v) mixture of chloroform and isoamyl alcohol. The isoamyl alcohol is added to help prevent foaming.
- The Phenol/Chloroform/Isoamyl Alcohol ratio is 25:24:1
- The most widely used method for concentrating DNA is precipitation with ethanol. The precipitate of nucleic acid, forms in the presence of moderate concentrations of monovalent cations (Salt, such as Na+), is recovered by centrifugation and redissolved in an appropriate buffer such as TE.
- The technique is rapid and is quantitative even with nanogram amounts of DNA.
- The four critical variables are the purity of the DNA, its molecular weight, its concentration, and the speed at which it is pelleted.
- DNA a concentrations as low as 20 ng/ml will form a precipitate that can be quantitatively recovered.
- Typically 2 volumes of ice cold ethanol are added to precipitate the DNA.
- Very short DNA molecules (<200 bp) are precipitated inefficiently by ethanol.
- Solutes that may be trapped in the precipitate may be removed by washing the DNA pellet with a solution of 70% ethanol. To make certain that no DNA is lost during washing, add 70% ethanol until the tube is 2/3 full. Vortex briefly, and recentrifuge. After the 70% ethanol wash, the pellet does not adhere tightly to the wall of thetube, so great care must be taken when removing the supernatant.
- Isopropanol (1 volume) may be used in place of ethanol (2 volumes) to precipitate DNA. Precipitation with isopropanol has the advantage that the volume of liquid to be centrifuged is smaller.