DNA Analysis

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DNA Review, Replication, Analysis
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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

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DNA is made of nucleotides (A, C, G, T) that
are anti-parallel
Replication/Transcription direction
Coding strand
Complimentary strand
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DNA molecular structure Make-up
3-OH
H-Bond
T
Transcription/ replication direction
A
C
G
T
A
Phosphate group
C
Complimentary strand
G
nucleotides
Deoxyribose sugar
3-OH
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Chromosomes1 from each parent
2 copies per cell
Nuclear DNA 3.2 billion bp
Located in cell nucleus
Autosomes
Sex-chromosomes
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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

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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)

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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

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RFLP

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

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RFLP
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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)

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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

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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

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PCR Process

• Heat DNA to 94C
• 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)

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

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What do STRs look like?
4,7 individual
5,5 individual
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How STRs Appear As a Result of Analysis
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CODIS
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FBI uses 13 different DNA loci153,581,500,000,00
0,000,000 probability
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The Same 13 Locus STR Profile in Different
Populations
1 in 837 trillion
1 in 0.84 quadrillion (1015) in U.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 in 18.0 quadrillion (1015) in U.S.
Hispanic population (NIST)
These values are for unrelated individuals
assuming 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
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CODIS

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