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DNA Analysis

DNA Analysis. DNA. DNA = D eoxyribo n ucleic A cid Located in CHROMOSOMES in the nucleus of cells What is a chromosome? Tightly packed genetic information Where do we get them from? One from each parent! Genes – portions of DNA that code for traits and functions. DNA. Timeline

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DNA Analysis

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  1. DNA Analysis

  2. DNA • DNA = Deoxyribonucleic Acid • Located in CHROMOSOMES in the nucleus of cells • What is a chromosome? • Tightly packed genetic information • Where do we get them from? • One from each parent! • Genes – portions of DNA that code for traits and functions

  3. DNA Timeline 1868 Miescher “discovers” DNA 1953 Watson and Crick report double helix structure 1977 First human gene cloned. 1984Jeffreysreports DNA sequences 1985 First report of PCR method 1986 Jeffreys uses DNA to solve first murder case 1987 First conviction on DNA evidence 1991 STRs first reported 1998 FBI starts CODIS database

  4. Discovery • Discovered in 1984 by Dr. Alec Jeffreys at the University of Leicester • He was knighted for his discovery

  5. 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

  6. 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 • 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

  7. What is DNA? • DNA is a POLYMER made up of nucleotides • Nucleotides are made up of three parts • Sugar (deoxyribose) • Phosphate • A nitrogen containing base (A,T,G,C) • There are approximately 100 million nucleotides in the average DNA molecule

  8. General Structure of DNA • Double helix—two coiled DNA strands • Composed of nucleotides connected together • Four bases make the rungs: • Adenine • Cytosine • Guanine • Thymine Bases always bind with specificity (complementary base pairing) A - T G – C

  9. Nucleotide A, G, C or T Forms sugar Phosphate Backbone What makes DNA Different from RNA?

  10. Phosphate Base Sugar Phosphate Base Sugar Phosphate Base Sugar Phosphate • Nucleic Acid Polymer (DNA) Polymer

  11. What is important about base pairs? • Can predict sequence of one strand based on the sequence of the other. • Responsible for Replication and Transcription • Every three nucleotides in DNA codes for one amino acid in the formation of a protein. • If a nucleotide is changed the “wrong” amino acid is placed in the protein and the protein may not function correctly and this is the basis for many diseases and health issues.

  12. Order in the Court! • The specific sequence of nucleotides of all human beings is 99.9% the SAME!! • It is that 0.1% difference that makes each person unique • What is the exception to this rule? Identical twins • What is so important about the sequence? It is the coding for proteins

  13. Match the sequence! • A T C G A C T A A C C G A C • T A G C T G A T T G G C T G

  14. Forensic Use of DNA • Forensic cases – matching suspects with evidence • Exonerate individuals • ID crime and catastrophe victims (ex. 9/11) • Establish paternity and other familial relations • Match organ donors with recipients in transplant programs • Missing persons investigations

  15. Bodily sources of DNA • All Cells that have a nucleus: • white blood cells (NOT RED BLOOD Cells-they don’t have a nucleus) • Semen • saliva –contains buccal (cheek cells) • hair root • Teeth • bone • Any tissue • All cells contains thousands of mitochondria(which contain maternal DNA)

  16. DNA “Typing” (Fingerprinting/Analysis) • 3 main technologies have been used: • RFLP –Restriction Fragment Length Polymorphisms - Developed in 1985 & used until mid 1990’s • PCR –Polymerase Chain Reaction • - Developed in the 1980’s, but perfected in mid 1990’s • - Still used today as a step in the STR process • STR-Short Tandem Repeats • - Developed in the 1990’s • - Used almost exclusively today

  17. The RFLP Technique • Restriction Fragment Length Polymorphism • Characterize fragments and calculate the statistical probability that two people could have the same fragment sequence • Portions of DNA contain sequences of bases that are repeated numerous times, known as tandem repeats. • These tandem repeats offer a means of distinguishing one individual from another. • They act as filler or spacers between the coding regions of DNA. • What is important to understand is that all humans have the same type of repeats, but there is tremendous variation in the number of repeats each of us have.

  18. Restriction enzymes cut DNA into fragments. They are thought of as highly specialized scissors that cut a DNA molecule when it recognizes a specific sequence of bases. • Length differences associated with relatively long repeating DNA strands are called restriction fragment length polymorphisms (RFLP) • Typically, a core sequence consists of 15 to 35 bases in length and repeats itself up to a thousand times.

  19. RFLP technique General Overview: 1)Isolate—separate DNA from the cell of desired individuals & evidence 2)Cut—using restriction enzymes to cut DNA into smaller fragments 3)Separate/ Sort—by size using electrophoresis which creates banding pattern. 4)Analyze—the specific allele patterns for identification

  20. DNA “Fingerprinting” RFLP • Restriction enzyme cuts at ….GAATTC….. • Spacing between cutting sites for enzyme is different for different individuals

  21. Analysis • Once the DNA molecules have been cut up by a restriction enzyme, the resulting fragments are sorted out by electrophoresis. • The smaller DNA fragments will move at a faster rate on the gel plate than the larger ones. • The fragments are then transferred to a nylon membrane • The nylon sheet is treated with radioactive probes to visualize the RFLPs

  22. RFLP: Electrophoresis • DNA is visualized using electrophoresis • Negatively charged DNA moves through a gel with a current • Smaller DNA moves faster than larger DNA fragments

  23. Which Suspect, A or B, cannot be excluded from the class of potential perpetrators of this assault?

  24. Three Possible Outcomes • Match —The DNA profile appears the same. • Excluded—The two samples originate from different sources. • Inconclusive—The data does not support a conclusion as to whether the profiles match.

  25. Down-side of RFLP • Need a relatively large amount of DNA • Large sequence size/lengths (do not allow for copying) • Requires a good sample (undegraded) • Relatively slow • Crime scene DNA is often in adverse conditions

  26. What to do when there’s not much there…

  27. PCR (Polymerase Chain Reaction) • Technique used for making copies of DNA molecule • PCR can amplify minute quantities of DNA many millions of times. • Uses high temperature enzyme that “mindlessly” copy DNA • After just 20 cycles a single copy of DNA is amplified over 2,000,000 fold. • Can be automated • Can use very small samples!

  28. PCR process - DNA “Fingerprinting” • Steps: • Isolate DNA piece • Heat/Denature: separation of DNA Strands (unzip) • Annealing: use primers [“start” and “stop” switches] to anneal [bind to complimentary DNA sequences] • Extension: use polymerases to build complimentary strand between “switches”

  29. A A T T G G C C A A T T T A T A A T A T G G C C DNA Amplification - PCR • Separated into 2 strands (Denaturation) • Heat or chemically

  30. T G G A A T C T G A G A G G A T A A A G T T T A C T A T T C T T A C T • Annealing- attaching primers (“start” and “stop” switches for DNA replication. • Extending- using polymerases Primer - fixed DNA binding points. A T G G A A A A T T T T T C C C T A T Completes first cycle - cycles repeated many times.

  31. PCR and RFLP • PCR technology cannot be applied to RFLP since the strands are too long (often numbering in the thousands of bases)

  32. Advantages of PCR • Typically 28-32 PCR cycles are run resulting in billions of copies of DNA • Each cycle that doubles the DNA takes about 2 minutes. • Minute amounts of DNA may be used for amplification (less than 1 billionth of a gram). • Can get enough DNA from envelopes, stamps, soda cans, & cigarette butts to run PCR

  33. Short Tandem Repeats (STRs) • The latest method of DNA typing uses short tandem repeat (STR)analysis. • STRs are locations on the chromosome that contain short sequences of 3-7 bases that repeat themselves within the DNA molecule (100-200x). • Serve as useful markers for identification since they are found in great abundance throughout the human genome • Because they are short, STRs are ideal candidates for multiplication by PCR • STRs are much less susceptible to degradation and may often be recovered from stains that have been subjected to decomposition

  34. By continuing the process with additional STRs from other genes, one can narrow down the probability of DNA belonging to a person.

  35. Short Tandem Repeats (STRs) STR typing is visualized by peaks shown on a graph. Each represents the size of the DNA fragment. The possible alleles are numbered for each loci. Norma's genotype is 15 repeats, 15 repeats at the locus D3S1358, 14 repeats, 16 repeats at vWA, 24 repeats, 25 repeats at FGA (gets on from each parent)

  36. Using STR DNA Profiling Results David & Karen are parents of a missing child: DNA Profile from remains found in a shallow grave: Could this be their child?

  37. Y- STR • Another tool available for crime scene investigations are STRs located on the Y chromosome, which is male specific. • More than 20 different Y-STR markers have been identified • Y-STRs will prove useful when multiple males are involved in a sexual assault or in question • The Y-STR is less complicated in appearance and interpretation.

  38. STR-PCR Advantages • Shorter DNA strands tend to be more stableand less susceptible to degradation • It can amplify minute quantities of DNA- need only 18 DNA bearing cells (100 times less than that normally required for RFLP analysis) • Works well on fragmented DNA • Morerapid analysis

  39. DNA Identification Based on Probability • Probability of a person matching a random DNA sample at any 1 STRS site is roughly 1/10 • 3 STRS sites? 1/10 x 1/10 x 1/10 = 1/1000 • All 13 STRS sites would mean that the chances of matching a random DNA sample are about 1 in 10 trillion: 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x = 1/10,000,000,000,000 • Probability of two different people matching at all 13 STRS sites is virtually zero.

  40. 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 Caucasians

  41. Where is DNA found? • Nuclear DNA • Located in the nucleus of cells • Inherited½ from mother and ½ from father • Mitochondrial DNA • Located in mitochondria (found in cytoplasm) • Inherited solely from mother • Used when nDNA typing is not possible (old, degraded samples) • More costly and time consuming

  42. Nuclear vs. Mitochondrial DNA

  43. Mitochondrial DNA • Shows maternal lineage (will be identical if related through the mother)

  44. FBI’s CODIS DNA Database Combined DNA Index System • Used for linking serial crimes and unsolved cases with repeat offenders • Launched October 1998 • Requires the 13 core STR markers • 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 crimeswith many states now expanding legislation to include other felonies • Forensic Profiles in NDIS (National DNA Index system – a component of CODIS): 119,782 • Convicted Offender Profiles in NDIS: 2,643,409

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