Cary Wong
DNA at crime scenes are usually subjected to harsh conditions that lead to
degradation and/or contamination. Fortunately, new analysis methods using
PCR amplification can accommodate degraded and very small samples of DNA.
One of these methods is analysis of short tandem repeats or STRs, which is
more sensitive than single-locus RFLP methods and less prone to allelic
dropout compared to VNTR methods.
Reducing the size of amplicons observed is another effective way to
minimize interference due to degradation. The reduced amplicons, called
miniSTRs, use primers located closer to the repeat regions, reducing the
size of the amplicon. One disadvantage of miniSTRs is the limited amount of
loci that can be amplified simultaneously.
The PCR process can be disrupted by PCR inhibition factors, leading to
incomplete amplification of samples. PCR inhibition can interfere with cell
lysis during DNA extraction, interfere by degrading nucleic acids present
and inhibit DNA polymerase activity. Solutions to PCR inhibition include:
diluting the DNA template (and the PCR inhibitor), using other polymerases,
and adding bovine serum albumin, sodium hydroxide or aluminum ammonium
sulfate.
Ch 8
Emmy Munoz
How many snps are needed? Most likely 50-100 is required to match the same
powers of discrimination and mixture resolution capabilities now achieved
with 10-16 STR loci. Large national and international efforts have been made
over the past years to catalog human variation found in the form of snp
markers. Mini sequensing involves alle-specific primer extension with
fluorescent dye-labeled dideoxynuceotide triphosphates to help visualize the
results. The three steps are amplification, primer extension and analysis.
Multiple primers can be analyzed simultaneously by linking a variable number
of additional nucleotides to the 5’end of the primer so that each primer
differs by several nucleotides from its neighbor. A major advantage of
allele-specific primer extension is that samples can be run on multi-color
fluorescence detection electrophorieses instruments. The technique is
sensitive and has the ability to be multiplexed and has proven to be fairly
robust. The applications are for the use to identify ethnic origins,
predicting physical traits of perpetrator and recovering more information
from degraded DNA sample. Snps have lower mutation rates than STRs and
therefore are more fixed in a population. The selling point of SNPs is the
ability to make small PCR products that can overcome challenges of strong
PCR inhibitors or sample possessing highly degraded material. Short
interspread nuclear elements are another form of repeated DNA that has been
investigated. SINEs have a short identifiable sequence inserted at a
location in the genome. Once inserted Alu elements are stable genetic marker
and are not subject to loss or rearrangement. Alu repeats have been shown to
yield information about geographic ethnic origins of the sample being
tested. Another is insertion-deletion it can be inserted or deleted a
segment of DNA ranging from one to hundreds of nucleotides. These markers
can be easily typed and may prove useful for future genetic studies.