Advantages and Limitations of DNA Typing Methods + DNA Quantification

rapid and simple tests

only test available for many years

blood group typing advantages

blood group typing limitations

poor power of discrimination with such few alleles (inclusions are not very meaningful)

protein profiling advantages

improved power of discrimination over blood group typing

poor power of discrimination even with multiple systems

poor sensitivity

proteins are not always stable in forensic stains or found in every sample

protein profiling limitations

excellent powers of discrimination

large numbers of alleles (20 to 30 bins) at each locus which facilitates mixed sample analysis

RFLP with single - locus VNTR probes advantages

limited sensitivity

time consuming process that cannot be automated

not suitable with degraded DNA samples due to high molecular weight needed

essentially continuous allele sizes which requires grouping alleles into bins

limited number of validated loci (4 to 6 loci commonly used) which meant that these VNTRs were of limited value in distinusing between siblings

RFLP with single - locus VNTR probes limitations

DQA1 + PM advantages

fast and simple method compared to RFLP

capable of analyzing small or degraded samples because it uses PCR

no instrumentation needed after PCR

DQA1 + PM limitations

poor power of discrimination with only six loci developed each containing only a few alleles

mixture interpretation difficult with limited number of alleles per locus

qPCR works exactly the same as PCR expect it adds two additional elements:

fluorescent dye

fluorometer

DNA template

DNA nucleotides (dNTPs)

taq polymerase

DNA primers

buffer

Ingredients of regular PCR:

Three main phases of PCR:

denaturing

annealing

extending

allows us to monitor the amplification process in real time

making it a quantitative process instead of a qualitative one

What makes qPCR different?

marks DNA nucleotides with fluorescent labels

sends out a signal when the dNTPs have been synthesized into new DNA

How does qPCR do this?