Diagnosis of Down syndrome by FISH

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

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

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

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

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

6. 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).

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

8. 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

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

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

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

14. 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.

15. 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.

16. 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.

17. DNA-based Tools STRs (Short Tandem Repeats)

18. 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.

19. C TP Th

20. Victim’s DNA Hair DNA

21. Allele frequency at TPOX locus CSF1PO allele 9: .02 Allele 10: .223 .02 X .223 X 2 = .009 THO1 allele 9.3: .345 .345 X .345 = .12 TPOX allele 11 .28 X .28 = .08 .08 X .12 X .009 = 8 X 10-5

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

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

24. 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

27. 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