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DNA Profiling PCR

DNA Profiling PCR. Some Phraseology Review. Recall from general biology the heirarchy of structure of DNA: Humans carry 2 copies of the DNA in their cells ( diploid ). The exception is sperm and eggs which contain one copy ( haploid )

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DNA Profiling PCR

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  1. DNA ProfilingPCR

  2. Some PhraseologyReview • Recall from general biology the heirarchy of structure of DNA: • Humans carry 2 copies of the DNA in their cells (diploid). The exception is sperm and eggs which contain one copy (haploid) • The DNA is organized into chromosomes – long strands of DNA • On the chromosomes, genes (sequences of DNA that code for a protein) are found. The location of the gene on the chromosome is its locus (plural: loci). • Much of the DNA is non-coding (junk DNA) and even in protein coding genes, there may be sequences that are cut out (introns) before they are used to make a protein. The remaining sequences are the exons. • Genes are sequences of DNA – there are only 4 building blocks of DNA (A,T,G and C), so the genes are actually sequences of these nucleotides. The length and order of nucleotides determines the type of protein that is produced by that gene. • Differences exist between individuals largely in the non-coding DNA (introns and junk DNA). DNA profiles detect and exploit these differences

  3. What is PCR?(basically a xerox machine) • DNA replication gone crazy in a tube! • Makes many copies of a specific target sequence from a small amount of template DNA • Affects gene mapping and cloning and DNA sequencing and detection • Applications in the detection of specific mutations, criminal investigations, and the human genome

  4. The Polymerase Chain Reaction • The polymerase chain reaction (PCR) produces many copies of a single gene or piece of DNA. • PCR requires DNA polymerase and a supply of nucleotides for the new DNA strands. • PCR is a chain reaction because the targeted DNA is repeatedly replicated as long as the process continues.

  5. The Polymerase Chain Reaction

  6. Amplifying the DNA of Interest • Because most tissue samples from a crime scene contain very little DNA, the goal is amplify, or make many copies of the DNA of interest • In STR analysis, you want to amplify the DNA containing the tandem repeats and only this DNA • The process used is called Polymerase Chain Reaction (PCR) • PCR Machines, or thermocyclers, use repeated cycles of heat and cooling to replicate the DNA using many of the same enzymes found in cells which facilitate DNA replication

  7. PCR • Ingredients: • 1. dNTPs (nucleotides) • 2. Buffer (to keep the pH and salt levels constant) • 3.Taq polymerase (heat stable DNA polymerase) • 4. Primers (short strands of DNA flanking the gene(s) of interest – they initiate DNA replication)

  8. PCR • Typical PCR reaction: • 1 minute 95 ºC to denature DNA (does what helicase does) • 1.5 minutes 60-65 ºC (allows primers to anneal) • 1 minute 72 ºC (allows Taq to add dNTPs) • This cycle is repeated 30-40 times produced millions of copies of the genes or sequences of interest

  9. Day 1 • Add Master Mix containing: • Nucleotides • Primers • Reaction buffer • Electrophoresis dyes • Taq polymerase Day 2 Day 3 PCR Procedures

  10. STR PCR (lots of acronyms) • The procedure is the same for STR analysis, but recall that each chromosome may have different numbers of STRs • The maternally and paternally inherited chromosomes usually have different numbers of inserts, so the result will be a 50/50 mix of amplified DNA with different repeats • For example, if you have 6 repeats from your mother and 2 from your father, you will amplify 2 different sized pieces of DNA – one larger than the other • In STR PCR, several different STR primers amplifying several areas of interest simultaneously

  11. How to amplify DNA...

  12. How to amplify DNA...

  13. Where does the data go? • CODIS – Combined DNA Index System

  14. CODIS • Uses 13 loci • All forensic laboratories that use the CODIS system can contribute to a national database. • Only Mississippi doesn’t participate • The Forensic Index contains DNA profiles from crime scene evidence. • The Offender Index contains DNA profiles of individuals convicted of sex offenses (and other violent crimes) with many states now expanding legislation to include other felonies. • Forensic Profiles in NDIS: 119,782 • Convicted Offender Profiles in NDIS: 2,643,409

  15. A Sample Profile • By combining the frequency information for all 13 CODIS loci, the frequency of this profile would be 1 in 7.7 quadrillion

  16. Case Study: The First Use of DNA Evidence • Two teenage girls raped and murdered in Leicestershire, England • Semen from the victims indicated a male with Type A blood and a rare enzyme = 10% of the local male population • A local boy, Richard Buckland, confesses upon interrogation • Police use DNA fingerprinting to confirm, but DNA profiles of Buckland and crime scene DNA do not match • Ironically, Buckland becomes the first person exonerated by DNA evidence

  17. Case Study: The First Use of DNA Evidence • Police request DNA samples from all adult males in 3 nearby villages (5000 men) • 6 months later – no results! • A year later, police are informed by a bakery worker that they overheard a co-worker bragging they had given a DNA sample for another man • Police obtain DNA from Colin Pitchfork and obtain a perfect match

  18. The Result? • In 1988, Colin Pitchfork was tried and convicted and sentenced to life in prison for the double rape and homicide based in large part to the DNA evidence

  19. As the technology gets smarter, so too do the criminals • A physician in Canada eludes authorities for years • Accused of drugging and sexually assaulting patients, DNA profiles from semen samples from the assaulted women do not match Dr. Schneeberger • Blood was drawn on 3 occasions in 1992, 1993 and 1996, but never came back as a match • Finally police obtain blood from a finger prick, swabbed the inside of his cheek and took hair samples • The results matched the DNA from the semen of the victims • How did he get away with it?

  20. As the technology gets smarter, so too do the criminals • On the previous 3 occasions, blood was drawn from the same arm • The last time the blood was drawn, the technician stated that the blood looked brown and “old” • Schneeberger had surgically implanted a piece of rubber tubing in his arm and filled it with stored blood from a patient

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