1. Chapter 2 DNA Biology Review ©2002 Academic Press

2. Target Region for PCR chromosome cell nucleus Double stranded DNA molecule Individual nucleotides DNA in the Cell ©2002 Academic Press

3. Basic Components of Nucleic Acids 5’end | Phosphate | Sugar—Base… | Phosphate | Sugar—Base… | 3’end Phosphate Sugar Base ©2002 Academic Press

4. Base Pairing of DNA Strands A = T G  C T = A A = T C  G T C C A G G T A G C T = A C  G A = T G  C 5’ 3’ 5’ 3’ 5’ 3’ 5’ denatured strands hybridized strands Hydrogen bonds C  G G  C Phosphate-sugar backbone ©2002 Academic Press

5. Basic Chromosome Structure p (short arm) centromere telomere q (long arm) telomere Band 5 Band 3 Chromosome 12 12p3 12q5 ©2002 Academic Press

6. http://www.ncbi.nlm.nih.gov/genome/guide/ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y Human Genome Contains 23 Pairs of Chromosomes Sex-chromosomes ©2002 Academic Press

7. TYPES OF DNA MARKERS Length Variation –short tandem repeats (STRs) microsatellites simple sequence repeats (SSRs) –minisatellites (VNTRs) Sequence Variation –single nucleotide polymorphisms (SNPs) –insertions/deletions (GATA)(GATA)(GATA)(GATA) (G/A) (CA)(CA)(CA)(CA(CA)(CA) ©2002 Academic Press

8. Family Inheritance of STR Alleles (D13S317) Father Child #1 Child #2 Child #3 Mother PCR product size (bp) 11 14 11 12 14 8 12 8 ©2002 AP, Elsevier

9. CharacteristicRFLP MethodsPCR Methods Time required to obtain results 6-8 weeks with radioactive probes; ~1 week with chemiluminescent probes 1-2 days Amount of DNA needed50-500 ng0.1-1 ng Condition of DNA neededhigh molecular weight, intact DNA may be highly degraded Capable of handling sample mixtures Yes (single locus probes) Yes Allele identificationBinning requiredDiscrete alleles obtained Power of Discrimination~1 in 1 billion with 6 loci~1 in 1 billion with 8- 13 loci (requires more loci) Comparison of RFLP and PCR ©2002 AP, Elsevier