Forensic Molecular GeneticsForensic Molecular GeneticsLecture 5Lecture 5

Ralph KirbyRalph Kirby

Amount of DNA extractable from a sample is Amount of DNA extractable from a sample is one of the main driving forces in the one of the main driving forces in the improvement of forensic DNA profilingimprovement of forensic DNA profilingThe second is the level of discriminationThe second is the level of discriminationThe third is the ease of explanation of the results The third is the ease of explanation of the results to a judge or a juryto a judge or a jury

RFLPRFLP

Easy to Easy to explainexplain

But needs But needs lost of DNAlost of DNA

Not very high Not very high discriminationdiscrimination

Polymerase Chain Polymerase Chain Reaction (PCR)Reaction (PCR)

Able to use very small Able to use very small quantities of DNAquantities of DNA

Amplifies specific Amplifies specific defined DNA sequencesdefined DNA sequences– Use specific short single Use specific short single

stranded DNA primersstranded DNA primers– Thermostable DNA Thermostable DNA

polymerase (Fidelity?)polymerase (Fidelity?)– Number of cyclesNumber of cycles– Detection systemDetection system

Contamination can Contamination can become a problembecome a problem

DQA systemDQA systemInvolves Specific Involves Specific genes and genes and specific alleles specific alleles available for such available for such genesgenesNeed to avoid gel Need to avoid gel electrophoresiselectrophoresisUses dot blotting Uses dot blotting (Southern (Southern Blotting)Blotting)Problem with Problem with similarity of similarity of markersmarkersUse of reverse Use of reverse dot blot with dot blot with internal controlinternal control

DQDQαα system in its system in its very early form very early form eliminated suspect 1 eliminated suspect 1 as the rapistas the rapistDQDQαα system as it system as it developed developed supported supported Quintanella as the Quintanella as the rapistrapistHowever, this still However, this still gave relatively poor gave relatively poor discrimination, <10% discrimination, <10% of the populationof the populationWould you convict Would you convict and send to jail for and send to jail for 99 years?99 years?

DQDQαα system as it became with 13 system as it became with 13 probes and 5 typed lociprobes and 5 typed loci

But hard to explain to a judge or juryBut hard to explain to a judge or jury

Need for an Need for an accurate but accurate but obvious system obvious system with good with good discriminationdiscriminationPCR amplification PCR amplification of Short Tandem of Short Tandem RepeatsRepeatsSeparated on an Separated on an automatic DNA automatic DNA sequencersequencerLabeled with Labeled with multiply coloured multiply coloured fluorescent dyesfluorescent dyesNeeds controlNeeds control

This is the type This is the type of control usedof control used

The STR system for The STR system for chromosomal markers is fine chromosomal markers is fine for DNA samples from most for DNA samples from most types of cases as they are not types of cases as they are not mixedmixedHowever, most DNA from rape However, most DNA from rape cases is a mixturecases is a mixtureCan separate sperm, but PCR Can separate sperm, but PCR still amplifies victims markersstill amplifies victims markersMixtures reduce discrimination Mixtures reduce discrimination and cause confusionand cause confusionHowever, in a rape case, only However, in a rape case, only Y chromosome that is present Y chromosome that is present in the sample will be from the in the sample will be from the rapist (Note possible exception rapist (Note possible exception of Y translocations and of Y translocations and in uteroin utero sex change)sex change)Thus STRs for Y chromosome Thus STRs for Y chromosome only have been developedonly have been developedQuite good discrimination, Quite good discrimination, especially if used in conjuction especially if used in conjuction with normal STRswith normal STRs

Extremely large Extremely large number of possible number of possible STR markers STR markers availableavailableMany commercial kits Many commercial kits availableavailableSome problems with Some problems with STRs and these will STRs and these will be discussed laterbe discussed later

But what about identification from skeletonsBut what about identification from skeletonsMitochondrial DNAMitochondrial DNA