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DNA Fingerprinting and Forensic Analysis

DNA Fingerprinting and Forensic Analysis. Chapter 13. PCR (Polymerase Chain Reaction). Technique for making copies or amplifying a specific sequence of DNA in a short period of time. Recombinant DNA. How is DNA Typing Performed?.

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DNA Fingerprinting and Forensic Analysis

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  1. DNA Fingerprinting and Forensic Analysis Chapter 13

  2. PCR (Polymerase Chain Reaction) Technique for making copies or amplifying a specific sequence of DNA in a short period of time.

  3. Recombinant DNA

  4. How is DNA Typing Performed? • Only one-tenth of 1% of DNA differs in each person; this variation can create a DNA profile of an individual • In criminal cases, DNA samples are obtained from crime scene evidence and a suspect and analyzed for the presence of a set of specific DNA regions

  5. How is DNA Typing Performed? • Portions of DNA contain sequences of letters that are repeated numerous times, called tandem repeats; act as filler or spacers between protein coding regions • All humans have the same type of repeats, but there is tremendous variation in the number of repeats that each of us have

  6. G C T G G T G C T G G C C T C

  7. How is DNA Typing Performed? • Two main types of forensic testing: • RFLP(Restriction Fragment Length Polymorphism) • PCR(Polymerase Chain Reaction) and STR (Short tandem repeats)

  8. DNA Typing • Restriction Fragment Length Polymorphisms • Sequence of 15 to 35 bases in length and repeats itself thousands of times • Each person will have a various number of repeated sequences • DNA cut by restriction enzymes; each person will have different fragments based on tandem repeats • Separate fragments by gel electrophoresis

  9. Restriction Fragment Length Polymorphisms • Person A -T A G A G A G A G A G-T A G A G A G A G A G Person B -T A G A G A G A G A G -T A G A G A G A G A G-T A G A G A G A G A G

  10. DNA Typing • RFLP • Requires a large amount of DNA • DNA must not be degraded • Crime scene evidence that is old or present in small amounts is often unsuitable

  11. DNA Typing • PCR and STR (Short Tandem Repeats) • Most successful and widely used DNA profiling • Locations in the DNA with SHORT sequences that repeat themselves • Each STR is normally 3-7 bases; only occurs in area of chromosome around 400 bases in length • Fragments created with restriction enzyme and multiplied by PCR • Fragments separated by electrophoresis

  12. Person 1 A A T G A A T G A A T G A A T G Person 2 A A T G A A T G A A T G A A T G A A T G A A T G

  13. DNA Typing • PCR • Requires less DNA • Still effective if the DNA is partially degraded • Extremely sensitive to contaminated DNA from the crime scene and within the laboratory

  14. How is DNA Typing Performed? • It is not necessary to catalog every base pair in an individual’s DNA profile to find the unique differences • DNA profiling depends on a small portion of the genome • Inactive, non-protein coding regions contain repeated sequences of base pairs called variable number tandem repeats (VNTRs)

  15. How is DNA Typing Performed? • VNTRs • Found in non-coding regions of chromosomes • Repeated sequences between 1 and 100 base pairs • Each person has some VNTRs inherited from each parent; therefore no person has VNTRs identical to either parent • VNTRs provides a scientific marker of identity known as a DNA fingerprint

  16. How is DNA Typing Performed? • VNTRs • Short repeated sequences, such as GTGTGT…….. • The number of repeats in each run varies in indivduals and are referred to as microsatellites or short tandem repeat

  17. Chapter 8.3 Preparing a DNA Fingerprint Specimen Collection • Investigators search a crime scene for any source of human cells! • Precautions must be taken to avoid possible contamination: • Wear disposable gloves and change frequently • Use disposable instruments when possible • Avoid talking, sneezing, and coughing to prevent contamination with micro-droplets of saliva • Avoiding touching any area that might contain DNA while handling evidence (face, nose, mouth, etc.) • Air-dry evidence thoroughly before packaging. Mold can contaminate

  18. Specimen Collection Enemies of evidence • Sunlight, high temperatures, and moisture can degrade DNA • Bacteria can contaminate samples

  19. Extracting DNA for Analysis • DNA can be extracted and purified • Chemically – using detergents to wash away unwanted cellular material • Mechanically – using pressure to force DNA out of a cell

  20. Restriction Fragment Length Polymorphism (RFLP) Analysis A. DNA is treated with restriction endonuclease (enzyme) to cut the DNA at specific points B. Use gel electrophoresis to separate the pieces B2. Gel is treated chemically to denature the DNA leaving two single strands; single stranded DNA is capable of binding to probes

  21. C. Southern Blot Technique i. Transfer fragments from gel to nitrocellulose or nylon membrane; the exact arrangement and position of the cut pieces of DNA is preserved on the transfer membrane (the “fingerprint”) ii. Membrane in incubated with a radioactive short strand of DNA called a probe; the probe contains the complementary sequence of bases iii. The binding of the DNA fragment with its complementary probe is called hybridization; after hybridization, any probe that does not bond with the DNA is washed away

  22. Southern Blot Analysis iv. X-ray (photo) film is placed on the nitrocellulose membrane; because the target DNA, with the probe attached, is radioactive and emits particles, an image forms on the photographic film. This is called can autoradiograph • Each DNA sample produces an image as bands located in specific positions; it is then possible to compare two or more autoradiographs to see if the bands match • RFLP is not used very much because it requires large amounts of DNA and can be easily contaminated http://www.dnalc.org/ddnalc/resources/shockwave/southan.html

  23. PCR and DNA Amplification a. Locate the portions of DNA that can be useful for comparison; primers are used to find these portions as they attach to the complementary DNA strand b. Once the complementary strand is located, copying begins using the thermal cycler (Refer to Chapter 3 notes to review the heating/cooling cycle to amplify DNA) c. PCR is sensitive to contamination and a small error in field or lab can result in the duplication of useless DNA

  24. D. Dot Blot (or Slot Blot) Analysis • Because the DNA produced by PCR is identical to the sample, gel electrophoresis is not needed to separate strands i. DNA is applied to specially prepared blot strips; each dot on the strip contains a different DNA probe ii. DNA probes in the “dots” are attached to an enzyme that can convert a colorless substrate (like DNA) into a colored one if binding occurs; if human DNA binds to its complementary probe, the dot changes color

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