DNA Fingerprinting

Use of DNA to Determine Identity DNA controls production of proteins DNA controls production of proteins Results in phenotype (eye color, facial features) Results in phenotype (eye color, facial features) Contributes to structure and function Contributes to structure and function DNA – 3 billion base pairs DNA – 3 billion base pairs No other person on planet has same code (except identical twins!) No other person on planet has same code (except identical twins!) Only 0.1% of DNA differs from person to person Only 0.1% of DNA differs from person to person But the regions that do vary provide a true genetic blueprint But the regions that do vary provide a true genetic blueprint

Use of DNA to Determine Identity DNA obtained from skeletal remains DNA purified If degraded – amplified w/ PCR RFLPs compared to determine final identity of missing person

What are some sources of DNA Evidence? Skin cells Skin cells Hair Hair Blood Blood Semen Semen Anything with DNA Anything with DNA

DNA Fingerprinting AKA - DNA profiling analysis or DNA typing AKA - DNA profiling analysis or DNA typing Electrophoretic analysis of DNA fragment sizes generated by restriction enzymes Electrophoretic analysis of DNA fragment sizes generated by restriction enzymes Provides accurate, unambiguous identification of source DNA samples Provides accurate, unambiguous identification of source DNA samples

Restriction Enzymes Endonucleases that cut phosphate bonds Endonucleases that cut phosphate bonds Break bonds between deoxyribose and phosphate Break bonds between deoxyribose and phosphate Attach to DNA and “read” nucleotides Attach to DNA and “read” nucleotides Cut at specific sequences Cut at specific sequences Over 3000 types Over 3000 types

Restriction Enzymes Named after the organisms from which they were discovered / isolated Named after the organisms from which they were discovered / isolated Eco RI – Escherichia coli RY13 Eco RI – Escherichia coli RY13 Hind III – Haemophilus influenzae R4 Hind III – Haemophilus influenzae R4 Bam HI – Bacillus amyloliquefaciens H Bam HI – Bacillus amyloliquefaciens H

Eco RI Function 5` - G A A T T C – 3` 3` - C T T A A G – 5` Eco RI 5`- GA A T T C – 3` 3` - C T T A A G – 5` DNA fragments with “sticky” ends

Restriction Enzymes Forensic labs – use at least 2 restriction enzymes Forensic labs – use at least 2 restriction enzymes 4-base and 5-base (sometimes others) 4-base and 5-base (sometimes others) Size of DNA fragments – depends on distance between recognition sites Size of DNA fragments – depends on distance between recognition sites Longer DNA molecule – the greater probability that a specific recognition site will occur Longer DNA molecule – the greater probability that a specific recognition site will occur

Restriction Enzymes Average human chromosome = 100 million bp Eco RI – 6 bp recognition site Probability – 1 site / 4096 bp Cut human DNA into ~25,000 fragments

Restriction Enzymes No two individuals have same pattern of restriction enzyme recognition sites No two individuals have same pattern of restriction enzyme recognition sites Each person – unique genotype (different alleles) Each person – unique genotype (different alleles) Mutations / Insertions / Deletions Mutations / Insertions / Deletions Changes distribution and frequency of restriction enzyme recognition sites Changes distribution and frequency of restriction enzyme recognition sites

After Restriction Digest Analyze DNA fragments on agarose gel Analyze DNA fragments on agarose gel DNA fragments separated by molecular weight (size) due to net negative charge on phosphates DNA fragments separated by molecular weight (size) due to net negative charge on phosphates Restriction enzyme cleavage of relatively small DNA molecules – key to RFLP analysis Restriction enzyme cleavage of relatively small DNA molecules – key to RFLP analysis If DNA fragments are too large – can’t see RFLP pattern If DNA fragments are too large – can’t see RFLP pattern

Sample RFLP Patterns Molecular Marker Control RFLP Suspect RFLP #1 Suspect RFLP #2 Suspect RFLP #3 Suspect RFLP #4 Suspect RFLP #5 Suspect RFLP #6

Restriction Enzyme Digestion Be sure to label all reaction tubes (4) Be sure to label all reaction tubes (4) 10 μl of enzyme reaction buffer 10 μl of enzyme reaction buffer 15 μl of DNA sample from missing person 15 μl of DNA sample from missing person 15 μl of Restriction Enzyme 15 μl of Restriction Enzyme Incubate at 37°C for ~60 minutes Incubate at 37°C for ~60 minutes Add 5 μl of Gel Loading Solution after incubation Add 5 μl of Gel Loading Solution after incubation