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DNA: Review, Replication, & Analysis

DNA: Review, Replication, & Analysis. Two types of DNA. Nucleic DNA Found in the nucleus of a cell Specific to an individual Mitochondrial DNA (mtDNA) Found in the mitochondria From the maternal side Not as specific – shows maternal side only. 3’. 5’. 5’. 3’.

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DNA: Review, Replication, & Analysis

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  1. DNA: Review, Replication, & Analysis

  2. Two types of DNA • Nucleic DNA • Found in the nucleus of a cell • Specific to an individual • Mitochondrial DNA (mtDNA) • Found in the mitochondria • From the maternal side • Not as specific – shows maternal side only

  3. 3’ 5’ 5’ 3’ DNA is made of nucleotides (A, C, G, & T) that are anti-parallel Replication/Transcription direction Coding strand Complimentary strand

  4. 5’- P 5’- P DNA molecular structure & Make-up 3’-OH H-Bond T Transcription/ replication direction A C G T A Phosphate group Complimentary strand C G nucleotides Deoxyribose sugar 3’-OH

  5. 2 copies per cell Chromosomes1 from each parent Nuclear DNA 3.2 billion bp Located in cell nucleus Autosomes Sex-chromosomes

  6. Chromosomes • Locus (pl. loci) specific location on the molecule (DNA) that contain: • Genes – unit of heredity • Made of different alleles • Homozygous = alleles are the same • Heterozygous = alleles are different

  7. Vocab Review • Recombinant DNA - opening up the base pr. Of the helix and recombining it with another strand • Restriction enzymes - chemicals that cut DNA into fragments that can later be incorporated into another DNA strand; ~150 different kinds • Polymer – long-chained molecule (e.g. DNA)

  8. Vocab Review • Probe - single strand of nucleic acid, much like RNA that has been made in a way that its base sequence lines up to hybridize areas on an allele; usually labeled w/ radioactive material • Polymerase - enzyme that is used to assemble new strands of DNA to the original/parent strand

  9. RFLP • Restriction Fragment Length Polymorphisms • Fragment lengths of repeating bases result from using restriction enzymes • 1st method used in forensic science

  10. RFLP

  11. RFLP Process • Need large amount of DNA • DNA is placed w/ restriction enzyme • Cut DNA is then separated using electrophoresis • DNA bands transferred to Nylon Membrane (Southern blotting) • Radioactive DNA probe is added to membrane (process called hybridization)

  12. RFLP Process (cont.) • X-ray film placed next to membrane for a couple of days • X-ray DNA film fragments then measured samples along with control (comparison) • RFLP strands used are typically thousands of bases long

  13. PCR DNA typing technique • Polymerase Chain Reaction • Now being used more than RFLP • Requires only small amount of DNA • Produces large amount of DNA • Can be used to aid other techniques • Uses electrophoresis • Best on strands no longer than a couple of hundred bases long

  14. PCR Process • Heat DNA to ~94°C • DNA becomes denatured • Annealing • Add primers (short strands of DNA) to separated strands • Primers combine or hybridize by lowering temp. • Add DNA polymerase (directs rebuilding of DNA strand) & mixture of free nucleotides. • Heat to ~55-72 °C • Repeat process 25-30 times. • This allows over 1 billion copies to be made (32 cycles)

  15. Simple Overview of PCR Amplification Denature, then anneal primers (short pieces of DNA to prime DNA synth) DNA synthesis Multiple cycles mg of DNA

  16. Short Tandem Repeats (STR) • Advantages • Better discrimination than RFLP • Faster result time • Low mutation rates • DNA has sections that repeat bases (2-7) these are used. • Uses capillary electrophoresis • Visualized as peaks on a graph

  17. What do STRs look like? 4,7 individual 5,5 individual

  18. How STRs Appear As a Result of Analysis

  19. CODIS

  20. FBI uses 13 different DNA loci1:53,581,500,000,000,000,000 probability

  21. The Same 13 Locus STR Profile in Different Populations 1 in 837 trillion 1 in 0.84 quadrillion (1015) inU.S. Caucasian population (NIST) 1 in 2.46 quadrillion (1015)in U.S. Caucasian population (FBI)* 1 in 1.86 quadrillion (1015)in Canadian Caucasian population* 1 in 16.6 quadrillion (1015)in African American population (NIST) 1 in 17.6 quadrillion (1015)in African American population (FBI)* 1 in18.0 quadrillion (1015)in U.S. Hispanic population (NIST) These values are for unrelated individualsassuming no population substructure (using only p2 and 2 pq) NIST study: Butler, J.M., et al. (2003) Allele frequencies for 15 autosomal STR loci on U.S. Caucasian, African American, and Hispanic populations. J. Forensic Sci. 48(4):908-911. (http://www.cstl.nist.gov/biotech/strbase/NISTpop.htm) *http://www.csfs.ca/pplus/profiler.htm

  22. CODIS • Combined DNA Index System • National DNA I.D. system • Has three levels • Local • State • Federal

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