مشخصات فایل:پاورپوینت روش های استخراج (DNA) از بدن
قالب بندی: پاورپوینت (قابل ویرایش)
تعداد اسلاید: 55
بخشی از پاورپوینت:DNA EXTRACTION METHODS
What are the essential components of a DNA extraction Procedure?
Maximize DNA recovery
Remove inhibitors
Remove or inhibit nucleases
Maximize the quality of DNA
Double strand vs. Single strand (RFLP or PCR)
How Much DNA Can We Recover?
A Diploid Cell contains approximately 6 pg of DNA
Sperm contains approximately 3 pg of DNA
The
average WBC of an adult is 5 - 10 X 106 cells per ml of blood.
Therefore, the theoretical recovery of DNA per ul of blood is 30 - 60
ng.
How Much DNA Do We Need?
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.
How Much DNA Do We Need?
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).
What are the Most Commonly used DNA Extraction Procedures in Forensic Science?
Organic (Phenol-Chloroform) Extraction
Non-Organic (Proteinase K and Salting out)
Chelex (Ion Exchange Resin) Extraction
FTA Paper (Collection, Storage, and Isolation)
ORGANIC EXTRACTION
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.
ORGANIC EXTRACTION PROCEDURE
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.
ORGANIC EXTRACTION REAGENTS
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.
ORGANIC EXTRACTION REAGENTS
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.
ORGANIC EXTRACTION REAGENTS
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.
ORGANIC EXTRACTION REAGENTS
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
Concentrating DNAAlcohol Precipitation
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.
Concentrating DNAAlcohol Precipitation
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.
Concentrating DNAAlcohol Precipitation
Very short DNA molecules (<200 bp) are precipitated inefficiently by ethanol.
The
optimum pelleting conditions depend on the DNA concentration.
Relatively vigorous microcentrifuge steps such as 15 minutes at or below
room temperature at 12,000 rpm are designed to minimized the loss of
DNA from samples with yields in the range of a few micrograms or less.
Concentrating DNAAlcohol Precipitation
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.
Concentrating DNAAlcohol Precipitation
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.
Isopropanol is less volatile
than ethanol and it is more difficult to remove the last traces;
moreover, solutes such sodium chloride are more easily coprecipitated
with DNA when isopropanol is used.
Concentrating DNAMicrocon®100 Centrifugal Filter Unit
Concentrating DNAMicrocon®100 Centrifugal Filter Unit
Excellent recovery of DNA samples with recoveries typically > 95%.
Ideal for dilute (ng/mL to µg/mL range) of DNA solutions
Concentrating and purifying proteins, antibodies and nucleic acids (alternative to EtOH precipitation)
Desalting and buffer exchange
Removal of primers, linkers and unincorporated label