Assay I HLA-DQ Alpha Haplotype

Purpose n To determine which one of several known alleles is present at the HLA DQ-Alpha locus on each of an individual’s two chromosomes n Why? u Tissue typing u Identifying genetic relatives u Suspect identification n Is this assay qualitative or quantitative?

Sample n Start with human hair follicle cells u Any human cells containing nuclear DNA n PCR the HLA DQ-Alpha region u PCR is an enzymatic technique used to increase the amount of sample

Detection Scheme n Probes - 8 oligonucleotides each with different sequence u Affixed to membrane strip u Sequence of each probe is that of a different HLA DQ-alpha allele

Detection Scheme n Specificity - complementary base pairing u Precise hybridization conditions limit pairing to exact complements F Hybridization = complementary base pairing of two single DNA or RNA strands to each other u PCR products from 1 person’s sample base pair with only 1 or 2 probes F 1 - homozygous F 2 - heterozygous

Visualization n Color reaction visible to naked eye u Color created by enzyme/substrate reaction F Enzyme = Horseradish peroxidase F Chromogen Substrate = TMB (tetramethylbenzidine) F Reaction = 2H 2 O 2 + TMB (colorless)  3H 2 O + oxidized TMB (blue)

How is the color localized to the Probe/PCR product complex? n PCR product includes biotin u Primers are biotinylated n Enzyme is conjugated to streptavidin u Conjugated = covalently bound n Streptavidin binds to biotin n So... enzyme/streptavidin conjugate localizes where PCR product is base-paired to probe n And...colored product is made and precipitates only where enzyme is

Contributions to sensitivity n Sample amplification by PCR n Signal amplification by repetitive enzymatic catalysis to yield more and more color n Background reduction u Blocking - prevents non-specific interactions u Rinsing - removes excess and misbound reagents n Note: Blocking and rinsing also contribute to specificity

Controls n PCR reaction u Positive control template – controls for failure of PCR reagents u Template negative – controls for presence of contaminant DNA u Agarose gel electrophoresis – checks for PCR yield n Hybridization u Control PCR product F Checks for hybridization success and specificity u Control probe on each strip F Warns of potential for false interpretation

Ask yourself n How has each of the essential assay components been incorporated into this assay?