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DNA Fingerprinting

DNA Fingerprinting. Sources of Biological Evidence. Blood Semen Saliva Urine Hair Teeth Bone Tissue. Characteristics of DNA. DNA is a polymer with repeating units called nucleotides . Each nucleotide has: sugar molecule phosphate group

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DNA Fingerprinting

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  1. DNA Fingerprinting

  2. Sources of Biological Evidence • Blood • Semen • Saliva • Urine • Hair • Teeth • Bone • Tissue

  3. Characteristics of DNA • DNA is a polymer with repeating units called nucleotides. • Each nucleotide has: sugar molecule phosphate group nitrogen containing group (base)

  4. Structure of DNA

  5. DNA is a double stranded helix • Two DNA strands come together to form a DNA molecule.

  6. Selective base pairing holds the strands together Adenine (A) only pairs with Thymine (T) Cytosine (C) only pairs with Guanine (G)

  7. Complementary Base Pairing • Bases can occur in any sequence on one strand. Opposite bases will be on the other strand. A T C C T G G C T T A T C G C T A G G A C C G A A T A G C G

  8. Where do proteins come from? • Proteins are made up of units called amino acids • Order of the amino acids determines the function and shape of the protein. • Triplet or three bases  amino acid C-G-T Alanine C-T-A Aspartate

  9. Normal and sickle cell red blood cells.

  10. Genetic diseases e.g. Sickle cell anemia: mutation in hemoglobin. Normal sequence: C C TG A GG A G Sickle cell anemia: C C TG T GG A G Changes the amino acid from glutamine  valine

  11. Human Genome Project Cost: $450 million (1990-2003) Aim: determine the order/sequence of bases on DNA contained in all 23 chromosomes. Why?

  12. DNA is “supercoiled”!

  13. Replication of DNA

  14. Replication of DNA • Parent strand must unwind. • Nucleotides are assembled to make new “daughter” strand. • Process continues until entire parent strand is copied. • End up with two exact copies.

  15. chromosome cell nucleus Double stranded DNA molecule Target Region for PCR Individual nucleotides DNA in the Cell

  16. Polymerase Chain Reaction DNA (e.g. crime scene, victim, suspect) + DNA polymerase + nucleotides (A, T, C, G) + primers Place in Thermal Cycler  multiple, exact copy of original DNA

  17. Original DNA target region Thermal cycle Thermal cycle Thermal cycle PCR Copies DNA Exponentially through Multiple Thermal Cycles In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created

  18. Polymerase Chain Reaction (PCR).

  19. Restriction enzymes • Restriction enzymes: “cut” DNA at specific sequences. e.g. Eco R1, Hind III, Pst I etc. EcoRI SmaI “sticky ends” “blunt ends”

  20. Recombinant DNA Technology Plasmid: vehicle used to transfer the “new” DNA into any cell.

  21. DNA Typing • Not all of DNA sequences code for the production of proteins • Tandem repeats can distinguish individuals.

  22. HLA DQ alpha system (DQA 1) • First commercial and validated PCR-based genetic marker system. • DNA typing of hair, saliva, semen stains etc.

  23. PCR produces multiple identical copies.

  24. How does PCR work? Consider, -G-C-T- T-C-C-A-G- -C-G-A-A-G-G-T-C- • Identify “primer sequences” and design primers. • Add DNA + primers + nucleotides (G,A,T,C) + DNA polymerase. • Heat DNA (separate the strands) • Cool DNA (primers anneal and DNA polymerase assembles new strand) • 1 CYCLE = two complete identical copies of DNA PCR animation (http://users.ugent.be/~avierstr/principles/pcrani.html)

  25. Short Tandem Repeats (STRs) • locations (loci)on the chromosome that have sequence elements that repeat themselves within the DNA molecule. • 3-7 bases in length, repeated many times. • Every person has 2 STR types for each element…..one inherited from each parent. • Analysis requires STR’s must be identified, number of repeats defined and sequence of bases flanking the STR

  26. STR Analysis and Multiplexing 1. Extract STR* (eg. THO1,ie. A-A-T-G) from biological sample. 2. Amplify by PCR 3. Separate on electrophoresis gel 4. Examine the distance moved to estimate to determine the number of STR repeats that exist. *Multiplexing allows simultaneous extraction and amplification of a number of different STRs

  27. Over 10 Markers Can Be Copied at Once Sensitivities to levels less than 1 ng of DNA Ability to Handle Mixtures and Degraded Samples Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges Multiplexing PCR

  28. Federal DNA Identification Act • 1994 • Combined DNA Index System (CODIS): facilitate the exchange of DNA typing data among police agencies investigating violent crimes and sexual assaults.

  29. DNA Index System • Mandated collection of : • Collection of DNA samples from convicted offenders • Establishment of DNA databases for law enforcement.

  30. Mitochondrial DNA. • Nuclear DNA (both parents) but mitochondrial DNA (maternal lineage only). • mtDNA is circular • mtDNA only contains information for 37 genes. • 2 regions (HV1 and HV2) have greatest number of variances.

  31. Collection and Preservation of Biological Evidence for DNA Analysis Photograph, notes, sketches Assume all body fluids are infectious Look for blood in less obvious places. Packaging! Refrigerate and store away from sunlight. DNA control samples.

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