molecular markers Flashcards
molecular markers:
a loose term to describe a specific piece of DNA that gives a unique piece of information. there are hundreds of different molecular markers used in forensic science.
they belong to distinct classes and are detected using different methods.
3 main markers- gene regions, SNPs, STRs.
gene regions:
an entire gene or part of a gene sequence. little variation in nuclear gene sequences as theyre coding. good for identifying different genes.
mtDNA gene sequences show more variation. good for identifying difference between species.
typically have to visualise the entire gene sequence to identify variable sites. each site represents a single nucleotide polymorphism.
genes of closely related individuals will be similar. can be used to track ancestry.
SNPs:
single nucleotide polymorphisms (SNPs). can occur in coding and non coding regions. some are highly informative.
SNPs occur through mutation events. most common at position 3 in triplet code.
STR:
STRs are co dominant. inherited in a mendelian fashion. 2 copies of an STR for each chromosome pair.
STRs can exist as a number of different repeat units.
heterozygote if fragments are different sizes.
homozygote if fragments are same size.
methods of detection: PCR
Polymerase chain reaction. basis of all DNA detection approaches. increases the number of copies of molecular marker. allows detection through fluorescence.
fluorescence detection:
DNA cant be observed directly. detection is done by measuring fluorescence. mechanism of fluorescence detection similar.
agarose gel:
most basic approach- detects genes (is primers are specific). detects different sized fragments.
DNA is negatively charged. DNA is run through a matrix (agarose). DNA is separated according to its relative mobility through the matrix.
small bands quickly travel. large bands travel slowly. precision is poor.
the agarose gel contains an intercalating dye. the dye binds in the groves of the double helix. the dye fluoresces in UV light when bound.
fragment length analysis:
same principles as agarose, sizing accuracy is better, can be used to detect STRs. uses a genetic analyser.
DNA sequencing:
same principle as FLA. uses a genetic analyser. requires detection of single base pairs. up to 800bp reads. detects SNPs and gene regions.
first PCR produces DNA fragment. second (sequencing) PCR adds fluorescent ddntp. results in multiple different terminal coloured dye.
qPCR:
combined the principles of PCR and agarose gel. fluorescence is detected as DNA is amplified. fluorescence is detected by using an intercalating dye. detects gene regions (is primers are specific). or molecular probe (taqman), detects gene regions as probe is highly specific to gene.
melt curve PCR:
uses the same PCR instrument. fluorescence is detected at the end of the PCR. detects gene regions and SNPs.
fluorescent probes bind to all the dsDNA that has been produced. the probe fluoresces when bound. the mix is then heated.
at a specific temperature the probe melts away from the DNA. a reduction in fluorescence is observed.
used in body fluid identification.
