DNA FINGERPRINTING:

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DNA FINGERPRINTING THE BASICS
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Look around the room.

• List 5 differences between yourself and others in
  the room.
• 1.
• 2.
• 3.
• 4.
• 5.

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DNA Fingerprinting

• Used for forensics, paternity testing, and
  identification of remains
• Small quantities of blood, semen or body tissue
  can be tested for DNA base sequences
• Each person has their own unique DNA fingerprint
  (nucleotide sequence) except for identical twins

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Most DNA is contained within the nucleus
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Some DNA is contained within the mitochondria of
the cell
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How to do DNA FingerprintingThe Big Picture
Collect Tissue Sample
gt20 cells
gt1000 cells
RFLP / Southern blot
PCR Analysis
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How does DNA fingerprinting work?

• Restriction enzymes cut DNA
• Pieces of DNA are run through a gel
  electrophoresis tray
• The DNA is probed with a labeled marker
• The pieces of DNA separate out by weight and make
  a banding pattern that is unique to an individual
  (fingerprint)
• Only about 10 regions are tested
• The probability of having matching DNA
  fingerprints is 1 in 1 million

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Step 1 Collect the tissue and isolate the DNA

• Lyse (break open) the cells
• Get rid of the proteins and lipids with organic
  solvent (e.g. phenol)
• Precipitate the nucleic acid with ethanol

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Step 2 Cut and run DNA out on a gel
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Restriction endonucleases aka Restriction enzymes

• Recognize specific base sequences in DNA
• Cut DNA at those recognition sites

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Step 3 Blot and probe gel- the Southern Blot
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Need a probe A short single stranded DNA which
is complementary to the region of interest


CAGTATACACAAGTACCGTACCTGGCTCAGTTATACGCCGA
A probe will base pair to the region of interest
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Variable Nucleotide Tandem Repeats (VNTRs)

• VNTRs are short segments of DNA that repeat a
  few to hundreds of times
• These unusual repeats occurs many different
  spots on human chromosomes
• Each individual will have different numbers of
  these VNTRs at each of these spots
• Each of these spots, or loci, are given
  different names (MSRs, STRs, AmpFLPs, etc)
• VNTRs are inheritable the numbers of repeats
  at each location in you are a random combination
  of the VNTRs in your parents

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RFLP Analysis

• RFLP Restriction Fragment Length
  Polymorphism for related DNA molecules, a
  difference in DNA fragment sizes after
  restriction enzyme digestion
• Difference results from presence of different DNA
  sequences
• Certain regions of genome are highly variable

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Simple Tandem Repeats(STRs)
STR region of DNA containing tandem copies of
di-, tri- or tetranucleotide repeat
units Examples Dinucleotide repeats
GTGTGTGTGTGT
Trinucleotide repeats ACGACGACGACG
Tetranucleotide repeats TATCTATCTATC
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• Number of repeats varies greatly between
  individuals
• STRs make up 10-15 of the mammalian genome
• STRs are also called microsatellites
• STRs are junk DNA
• Y-STRs- STRs specific for the Y chromosome

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PCR
Purpose Quickly make many copies of a region
of a DNA molecule Method Multiple rounds of
DNA replication Components in PCR reaction
Target DNA, nucleotides, DNA polymerase, and
primers
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PCR to Amplify a Persons DNA

• Steps Involved
• Isolate a persons
• Design primers

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• Add vast excess of the primers and heat mixture
  to 75 oC
• This causes DNA strands to separate by breaking
  hydrogen bonds between bases

4. Cool to 15 oC. Primers hydrogen bond (anneal)
to complementary strands
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• Add DNA polymerase and all four types of
  nucleotides. The polymerase (enzyme used in DNA
  replication) will fill in the rest of the two
  strands.
• You now have two identical copies of the DNA you
  started with.

6. REPEAT !!!!!!
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If all RFLP and STR regions are considered, there
is a one in 3.4 billion chance of error. This
means there may be one other person on the planet
that would be too similar to tell the
difference. If other VNTR regions are also
considered, the chances of error go way, way
down Clearly, suspect one is the match..