DNA DNA FingerprintingFingerprinting

Use of DNA to Determine Use of DNA to Determine IdentityIdentity

DNA controls production of proteinsDNA controls production of proteins Results in phenotype (eye color, facial Results in phenotype (eye color, facial

features)features) Contributes to structure and function Contributes to structure and function

DNA – 3 billion base pairsDNA – 3 billion base pairs No other person on planet has same code No other person on planet has same code

(except identical twins!)(except identical twins!) Only 0.1% of DNA differs from person to Only 0.1% of DNA differs from person to

personperson But the regions that do vary provide a true But the regions that do vary provide a true

genetic blueprintgenetic blueprint

Use of DNA to Determine Use of DNA to Determine IdentityIdentity

DNA obtained from DNA obtained from skeletal remainsskeletal remains

DNA purifiedDNA purified If degraded – If degraded –

amplified w/ PCRamplified w/ PCR

RFLPs compared to RFLPs compared to determine final determine final identity of missing identity of missing personperson

What are some sources of What are some sources of DNA Evidence?DNA Evidence?

Skin cellsSkin cells

HairHair

BloodBlood

SemenSemen

Anything with DNAAnything with DNA

DNA FingerprintingDNA Fingerprinting

AKA - DNA profiling analysis or DNA AKA - DNA profiling analysis or DNA typingtyping

Electrophoretic analysis of DNA Electrophoretic analysis of DNA fragment sizes generated by fragment sizes generated by restriction enzymesrestriction enzymes

Provides accurate, unambiguous Provides accurate, unambiguous identification of source DNA samplesidentification of source DNA samples

Restriction EnzymesRestriction Enzymes Endonucleases that Endonucleases that

cut phosphate bondscut phosphate bonds Break bonds between Break bonds between

deoxyribose and deoxyribose and phosphatephosphate

Attach to DNA and Attach to DNA and “read” nucleotides“read” nucleotides

Cut at specific Cut at specific sequencessequences

Over 3000 typesOver 3000 types

Restriction EnzymesRestriction Enzymes

Named after the organisms from Named after the organisms from which they were discovered / isolatedwhich they were discovered / isolated

Eco Eco RI – RI – Escherichia coliEscherichia coli RY13 RY13

HindHind III – III – Haemophilus influenzaeHaemophilus influenzae R4 R4

BamBam HI – HI – Bacillus amyloliquefaciensBacillus amyloliquefaciens HH

EcoEco RI Function RI Function

5` - G A A T T C – 3`3` - C T T A A G – 5`

Eco RI

5`- G A A T T C – 3`3` - C T T A A G – 5`

DNA fragments with “sticky” ends

Restriction EnzymesRestriction Enzymes

Forensic labs – use at least 2 restriction Forensic labs – use at least 2 restriction enzymesenzymes 4-base and 5-base (sometimes others)4-base and 5-base (sometimes others)

Size of DNA fragments – depends on Size of DNA fragments – depends on distance between recognition sitesdistance between recognition sites

Longer DNA molecule – the greater Longer DNA molecule – the greater probability that a specific recognition site probability that a specific recognition site will occurwill occur

Restriction EnzymesRestriction Enzymes Average human Average human

chromosome = 100 million chromosome = 100 million bpbp

Eco Eco RI – 6 bp recognition RI – 6 bp recognition sitesite

Probability – 1 site / 4096 Probability – 1 site / 4096 bpbp

Cut human DNA into Cut human DNA into ~25,000 fragments~25,000 fragments

Restriction EnzymesRestriction Enzymes

No two individuals have same pattern of No two individuals have same pattern of restriction enzyme recognition sitesrestriction enzyme recognition sites

Each person – unique genotype (different Each person – unique genotype (different alleles)alleles)

Mutations / Insertions / DeletionsMutations / Insertions / Deletions Changes distribution and frequency of Changes distribution and frequency of

restriction enzyme recognition sitesrestriction enzyme recognition sites

After Restriction DigestAfter Restriction Digest Analyze DNA fragments on agarose gelAnalyze DNA fragments on agarose gel

DNA fragments separated by molecular DNA fragments separated by molecular weight (size) due to net negative charge on weight (size) due to net negative charge on phosphatesphosphates

Restriction enzyme cleavage of relatively Restriction enzyme cleavage of relatively small DNA molecules – small DNA molecules – key to RFLP key to RFLP analysisanalysis

If DNA fragments are too large – can’t see If DNA fragments are too large – can’t see RFLP patternRFLP pattern

Sample RFLP PatternsSample RFLP PatternsM

olec

ular

Mar

ker

Contr

ol

RFLP

Susp

ect R

FLP #

1

Susp

ect R

FLP #

2

Susp

ect R

FLP #

3

Susp

ect R

FLP #

4

Susp

ect R

FLP

#5

Susp

ect RFL

P #6

Restriction Enzyme Restriction Enzyme DigestionDigestion

Be sure to label all reaction tubes (4)Be sure to label all reaction tubes (4) 10 10 μμl of enzyme reaction bufferl of enzyme reaction buffer 15 15 μμl of DNA sample from missing personl of DNA sample from missing person 15 15 μμl of Restriction Enzymel of Restriction Enzyme

Incubate at 37°C for ~60 minutesIncubate at 37°C for ~60 minutes

Add 5 Add 5 μμl of Gel Loading Solution after l of Gel Loading Solution after incubationincubation