Diagnosis of Down syndrome by FISH Chromosome 13 probe Chromosome 21 probe

Nucleic acid Basics Hybridization Electrophoresis PCR Diagnostic tools DNA-Protein interactions Chromatin Gene expression

3’ 5’ Single stranded DNA 5’ 3’ Primer (~20 nucleotides long) PCR DNA polymerase can extend a primer DNA polymerase dATP, dGTP, dCTP, dTTP 3’ 5’ 5’ 3’

3’ 5’ PCR DNA polymerase MUST have a primer DNA polymerase dATP, dGTP, dCTP, dTTP No primer, no reaction 3’ 5’ Single stranded DNA

3’ 5’ 5’ 3’ Denatured DNA strand 3’ 5’ Denatured DNA strand 5’ 3’ Primers (~20 nucleotides long) PCR + DNA polymerase + dATP, dGTP, dCTP, dTTP heat cool Heat and cool 20 times Convince yourself that this will be nearly the sole product

DNA-based Tools PCR –Excellent for amplification. –Good for detecting mutations that result in small changes in allele size. –There is a limit to the size of the PCR product (about 500 bp) –Semiquantitative (can not distinguish one copy of a gene from two copies of the gene).

Nucleic acid Basics Hybridization Electrophoresis PCR Diagnostic tools DNA-Protein interactions Chromatin Gene expression

DNA-based Tools AMPLIFICATION BY PCR Detection of Infectious agents –Commonly used for detection of clamydia and gonorrhea Obtain sample; prepare DNA PCR amplify with primers specific for DNA of organism Measure the amount of DNA produced by the PCR reactions

DNA-based Tools Mutation detection by PCR Detection of small deletions or insertions –Example: detection of the deltaF508 cystic fibrosis conductance regulator (CFTR) allele

Nucleic acid Basics Hybridization Electrophoresis PCR Diagnostic tools DNA-Protein interactions Chromatin Gene expression

DNA-based Tools STRs (Short Tamdem Repeats) (Also called SSR (simple sequence repeat) or microsatellite) –Paternity testing –Forensics –Detection of microsatellite instability

DNA-based Tools STRs (Short Tandem Repeats) On September 15, 1990, near the city of Prague, the body of a woman was discovered in a trench. The identity of the victim was soon established. The cause of death was determined to be the result of ligature strangulation.

DNA-based Tools STRs (Short Tandem Repeats) Between January 1991 and April 1992 the bodies of seven prostitutes were discovered in wooded areas in different parts of Austria. In June and July 1991, the bodies of three prostitutes were discovered in Los Angeles.

DNA-based Tools STRs (Short Tandem Repeats) In May 1991, a potential suspect for the murders in Austria was identified. Suspicious looking hair was found in the suspect’s car.

DNA-based Tools STRs (Short Tandem Repeats)

DNA-based Tools STRs (Short Tandem Repeats) Two nanograms of DNA was obtained from hair found in the suspect’s car. PCR was used to examine several STR loci.

C TP Th

Victim’s DNA Hair DNA

Allele frequency at TPOX locus TPOX allele X.28 =.08 CSF1PO allele 9:.02 Allele 10: X.223 X 2 =.009 THO1 allele 9.3: X.345 = X.12 X.009 = 8 X 10-5

DNA-based Tools STRs (Short Tandem Repeats) The suspect was convicted and is currently serving a life sentence in Austria.

Nucleic acid Basics Hybridization Electrophoresis PCR Diagnostic tools DNA-Protein interactions Chromatin Gene expression

DNA-based Tools ASO (Allele Specific Oligonucleotides) –Used to detect specific alleles. –Can determine if a person is homozygous or heterozygous for a particular allele

DNA-based Tools ASO (Allele Specific Oligonucleotides) –Good Knowledge of the allele to be tested is required for distinguishing one allele from another –Usually used in conjunction with PCR, electrophoresis and DNA blotting