Quantitative PCR
Real Time PCR
-Detection and quantitation of fluorescent reporter the signal of which increases in direct proportion to the amount of PCR product in a reaction
-Does not measure the amount of end product but its production in real time
SYBR green
Also bindsprimer dimers
Can overestimate product
Molecular Beacons
-Uses FRET
-Fuorescence Resonance Energy Transfer
-Uses two sequence specific oligonucleotides labeled with fluorescent dyes
Taq Man
Other application of PCR
-Detection of mutationsscreen for inherited disorders
-Detection of HIV
-Not standard test given
-Detect tuberculosis without culturing
-Prenatal sex determination
-DZY1 = Y specific sequence present in 5000 copies
-Preimplantation diagnosis of genetic diseases
-Forensics
-Paternity testing
Forensics
STR
Short Tandem Repeats
2 to 7 base pairs repeated 7-40 times
Replaced VNTRs in forensic analysis
13 Highly polymorphic loci have beenselected by FBI
Population match probabilities 0.1 - 0.28
Probability One in 5.7 X 10-15
Combined DNA Index System (CODIS)
STR analysis of family of last Tsar of Russia
VNTR’s
Can use PCR to visualize VNTRs
Eg. pMCT118 in chromosome 1
VNTR analysis
Problems with PCR
Contamination
Theoretically one molecule
Takes one mismatchthe wrong fragment
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