DNA Extraction methods

Critique of Organic extraction

• https://www.youtube.com/watch?v=JNl1kjw9ZDQ
• it is time consuming
• involves the use of hazardous chemicals,
• requires the sample to be transferred among
multiple tubes, increasing risk of error and
contamination.

• works well for recovery of high-molecular-weight

double stranded DNA
• Suitable for DNA to be used in STR/RFLP analyses
 Chelex
• Contains chelating groups that bind polyvalent metal
ions such as magnesium.

– DNA-destroying nuclease enzymes are inactivated and the

DNA molecules are thus protected.

• denatures the DNA as well as disrupt the cell

membranes and destroy the cell proteins.

• centrifugation pulls the Chelex resin and cellular

debris to the bottom of the tube, the supernatant is
removed and can be used directly for PCR
 Critique of Chelex
• Removes inhibitors of PCR
• uses only a single tube for the DNA extraction,
which reduces the potential for laboratory-
induced contamination.
• Method is inexpensive
• not effective for RFLP typing because Chelex
denatures double-stranded, producing single
stranded DNA (Chelex non English)
• Chelex part 1 and Chelex part 2
 FTA Paper
• Preserves the DNA sample from nucleases and
bacterial growth
• consistent results may be obtained without
quantification.
• The procedure may be automated on a robotic
workstation.
• Unfortunately they jump about
• The basic premise of purifying DNA using FTA®
paper is simple: biological samples are applied to
the FTA® paper and air-dried.

• A small disc of the FTA® paper is then removed,

and washed to remove any non-DNA material
(the DNA remains entangled within the paper).

• Analysis can subsequently be performed on the

DNA whilst still attached to the paper, or the
DNA can be eluted prior to use.
 Solid-Phase DNA extraction
• DNA is selectively bound to a substrate such as
silica particles and then released following
stringent washes that purify the bound DNA
molecules
• In the Qiagen System DNA selectively adsorb to a
silica support, such as small glass beads, in the
presence of high concentrations of chaotropic
salts such as guanidine hydrochloride, guanidine
isothiocyanate, sodium iodide
• If the solution is less than pH 7.5, DNA adsorption
to the silica is typically around 95% and unwanted
impurities can be washed away.

• Under alkaline conditions and low salt

concentrations, the DNA elutes from the silica
• Qiagen DNA extraction
• Others solid-phase systems include: DNA IQ by
PROMEGA and Prepfiler – ABI
 Differential Extraction
• Used to isolate the female and male fractions in sexual
assault cases that contain a mixture of male and female
DNA.
• By separating the male fraction away from the victim’s DNA
profile, it is much easier to profile the perpetrator’s DNA

• Involves breaking open the female epithelial cells with

incubation in an SDS/ proteinase K mixture.

• Sperm nuclei are subsequently lysed by treatment with an

SDS/ proteinase K/ dithiothreitol (DTT) mixture. The DTT
breaks down the protein disulfide bridges that make up
sperm nuclear membranes.
• Method for directly capturing sperms cells have
been developed including laser capture
microdissection

• When sperm cells are observed in the field of

view of the microscope, a tiny laser is activated

• a thin plastic film placed over the slide melts at

the specific point of laser light contact to
capture or embalm the cell of interest.
 Inhibitors of PCR

• Common ones encountered: melanin, indigo

dye, heamoglobin

• Act by binding to Taq polymerase

 Degraded DNA
• Nucleases, bacteria, fungi, and insects all degrade DNA

• hydrolytic cleavage (water)

• oxidative base damage

• UV irradiation (e.g., direct sunlight) can lead to

crosslinking of adjacent thymidine nucleotides on the
DNA molecule, preventing polymerase working

•
 Quantitation of the DNA
• Necessary for most polymerase chain reaction
(PCR)-based assays as a narrow concentration range
works best with multiplex short tandem repeat (STR)
typing.

– Between 0.5 and 2ng of DNA

– Too much DNA causes the profile to be ‘overblown’

– Too little DNA can result in loss of alleles due to stochastic

amplification and failure to equally sample the STR alleles
present in the sample.
 Normalization
Before amplification samples must be
normalized:

• Samples too dilute must be concentrated

• Samples too concentrated must be diluted
Stochastic effects when low DNA and
many PCR cycles
 UV absorbance and fluorescence
• Early methods of quantitation typically
involved either measurement of absorbance
at a wavelength of 260 nm or fluorescence
after staining a yield gel with ethidium
bromide.

• Methods not sensitive

• Absorbance not specific to DNA (phenols and
proteins)
 Slot Blot
• Used in the 1990s
• Specific to human and DNA of higher primates

• Used a 40bp probe that bound to D17Z1 on

chromosome 17

• DNA to be quantified was immobilized on a nylon

membrane then washed with probe

• Used radioactive, chemiluminescent and

colorimetric detection which were compared to
standards
 Critique of slot blot

• Consumed some of DNA sample

• If comparison done visually, results subjective

• relatively sensitive (150 pg DNA could be detected)

• Obsolete

• Not specific to human

 PicoGreen microtiter plate assay

• PicoGreen is a fluorescent. interchelating dye whose

fluorescence is greatly enhanced when bound to
double-stranded DNA
• Very sensitive
• Uses a 96 well microtiter plate, quantifies 96 samples
at a time.
• Detection achieved with a fluorometer
• Sample is compared to a standard curve
• Process automated
• Not specific to Human DNA
 Aluquant human DNA quantitation
system
• detection of DNA using Alu repeats that are in
high abundance in the human genome.
• Probe-target hybridization initiated a series of
enzymatic reactions that ended in oxidation of
luciferin with production of light.
• Light detected using a luminometer and
compared to a standard curve
 critique
• Measured 0.1-50ng DNA- Fairly sensitive

• Specific to human DNA

• Automated process

• Now obsolete
 Real time PCR

• Gives information on quality and quantity of

DNA
• it analyzes the cycle-to-cycle change in
fluorescence signal resulting from
amplification of a target sequence during PCR.
• Real time PCR
• Visual Rep of Q Pcr