EDVOTEK 225 DNA Fingerprinting

Report
EDVOTEK 225
DNA Fingerprinting
Spring 2010
Terry Kotrla, MS, MT(ASCP)BB
DNA Fingerprinting
• Also called
– DNA profile analysis
– DNA typing
• Involves electrophoretic anlaysis of DNA
fragment sizes generated by restriction
enzymes
• More accurate an unambiguous then blood
typing
Restriction Enzymes
• Restriction enzymes are endonucleases that
catalyze cleavage of phosphate bonds
• Require Mg-2 for activity
• Generate 5’ phosphae and 3’ hydroxyl group
• Endonuclease claves at specific sequence of
bases.
• Produce by bacteria
Restriction Enzymes
• Names after organism discovered in.
– First 3 letters of genus
– Strain or substrain
– Roman numeral to designate one of the enzymes
produced by the same organism.
Restriction Enzymes
• Require a specific double stranded sequence
of nuceotide bases to cut DNA
• Recognition sites usually 4-8 base pairs within
or neat site
• Frequently symmetrical, both DNA strands
have same base sequence when read 5’ to 3’,
ie, palindrome.
• In forensics 2 most commonly used are Hae III
and Hinf I.
RFLP
• Restriction Fragment Length Polymorphisms
– http://tinyurl.com/2evjhfu
Restriction Enzymes
• Size of fragments depends on distances
between recognition sites.
• Long DNA molecule will have higher
probability recognition site will be present.
• Restriction enzyme with 6 bp recognition site
expected to cut human DNA into
approximately 750,000 different fragments
Fragments
• No 2 individuals have same pattern of
recognition sites.
– Alleles result in alternative expressions of genetic
traits, dominant or recessive
– Two copies of gene at given locus, mom & dad
– Alleles have differences in base sequences
– Mutations and deletions occur which eliminate
palindromic site (figure 2)
Repeats
•
•
•
•
Repetitive base sequences occur
Constitute large fraction of mammalian genome
Have no known genetic function
10-15% of DNA consists of repeated short
sequences.
• Vary between individuals
• When flanked by recognition sites the length of
repeat will determine size of fragment generated.
Electrophoresis
• Used to analyze DNA fragments
• DNA has negative charge.
• Gel is a sieve which separate DNA fragments
according to size, charge and shape.
• Only size of DNA affects mobility.
• Cleavage of large complex human DNA generates
fragments which may exceed resolving capacity
of gel.
• Cleaved DNA will appear as a smear, no bands.
Southern Blot
• Electrophoresis is done.
• Denature DNA fragments in alakali solution.
• Double stranded fragments converted to
single stranded form.
• Trnafer DNA fragments to nitrocellulose
(blotting)
• Blotted DNA hybridized with labeled
oligonucleotide DNA probe.
Southern Blot
• Probe
– DNA fragment which is complementary to
sequences found on human chromosomes
– Labeled with a “reporter” to detect target
– Probe incubated with blotted membrane and will
hybridize to complementary sequences on
membrane.
– Allows only specific DNA fragments to be
detected.
Forensics
• DNA samples collected at crime scene extracted from
–
–
–
–
Skin
Blood
Semen
Hair
• Perform RFLP analysis
• If pattern matches it will prove beyond a reasonable
doubt that suspect was at crime scene.
• Forensics uses different sets of probes to hybridize to
different repetitious sequences to satisfy statistical
probability for positive identification.
DNA and PCR
• PCR very sensitive, requires very little DNA
• Allows increased speed for analyzing
• Requires
– DNA polymerase Taq polymerase
– Four dinucleotides
– Primers - two synthetic oligonuceotides 15-30 bp
in length which correspond to start and end of
DNA to be amplified (target)
– Buffer with Mg2+
PCR
• Three phases
– Heat to 92-96C to denature DNA
– Cool to 45-65 to allow primers to anneal to
separated strands
– Heat to 72C to all extension, nucleotides added to
primers.
DNA Fingerprinting
• Significant in court cases such as:
– Murder
– Rape
– Physical battery
• Results in acquittal or conviction
• Process is standardized
Procedure
• PCR has been done
• Have crime scene DNA and 2 suspects
• Will treat all 3 samples with 2 restriction
enzymes.
• After enzyme treatment will electrophorese
and stain.
• Analyze gels to determine guilty suspect, one
whose DNA matches crime scene DNA

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