Chapter 13

1. Chapter 13 DNA: The Indispensable Forensic Science Tool

2. Objectives: • Describe and demonstrate DNA collection, preservation, and testing, using gel electrophoresis. • Use Product Rule to quantify and interpret results.

3. Father of DNA fingerprinting • Alec Jeffreys • 1985 • Responsible for the revelation of DNA fingerprinting

4. DNA fingerprinting • Isolating and reading DNA markers (portions of the DNA structure of certain genes that are unique to each individual).

5. What is a gene? • Fundamental unit of heredity. • Instructs body cells to make proteins. • The proteins are responsible for determining specific traits.

6. What is DNA? • Polymer that carries the body’s genetic information. • Double helix. • Watson & Crick – built ball & stick model of DNA.

7. What is a polymer? • Large molecule that links together a series of repeating units. • In DNA, the repeating units are called nucleotides.

8. What are nucleotides? • Building blocks of DNA molecule. • Composed of • Sugar (backbone) • Phosphate group (backbone) • Nitrogen base.

9. 4 Nitrogen Bases in DNA • Adenine + Thymine = complementary bases. • Cytosine + Guanine = complementary bases • Three nitrogen bases are called a codon. • Each codon codes for a specific amino acid. • Groups of amino acids produce a protein.

10. DNA to Protein • The specific proteins produced by a cell are directly related to the sequence of nucleotides in the DNA of the cell. • The individuality of an organism is determined by the organism’s DNA nucleotide sequence. • Sickle cell anemia (example)- abnormal hemoglobin

11. Human Genome • Total DNA content found within the nucleus of a cell.

12. Human Genome Project • Global effort designed to reveal the location of all genes in the human genome. • Will be useful in diagnosing & treating genetic diseases. • Will help reveal the role & implications of evolution.

13. DNA Replication • Synthesis of new DNA from existing DNA. • Semiconservative.

14. Steps of DNA Replication • Helicase • Enzyme that holds double strands apart (forks) • DNA Primer • Attracts DNA polymerase • DNA Polymerase • Enzyme that inserts complementary base pair & proofreads the new DNA strand for errors. • Ligase • Enzyme that seals the new strand to the old strand of DNA.

15. PCR • Polymerase Chain Reaction. • Technique for copying a particular DNA portion of interest outside a living cell. • Can produce many exact copies of segments of DNA so sample size is no longer a limitation of DNA physical evidence. • 1st step is to heat the DNA strands to cause them to separate completely. • Each cycle of the DNA Thermal Cycler takes approximately 2 minutes.

16. Recombinant DNA • The cutting of DNA into fragments that can later be incorporated into another DNA strand. • Enormous commercial implications (ex: growth hormones, insulin, etc)

17. Restriction Enzymes • Chemicals that act as scissors to cut DNA molecules at specific locations. • Produces RFLPs.

18. RFLPs • Restriction Fragment Length Polymorphism • Different fragment lengths of base pairs that result from cutting a DNA molecule. • All humans have the same type of repeats, but vary in the number. • Typically a core repeat = 15-35 bases. • Radioactive probes are used to visualize RFLPs (make them gel visible).

19. Tandem Repeats • Region of a chromosome that contains multiple copies of a core DNA sequence that are arranged in a repeating fashion. • Origin & significance is a mystery. • About 30% of human genome is largely composed of repeating segments of DNA. • It is thought that they may act as spacers between the coded regions of DNA.

20. Steps of DNA Typing • Digestion with restriction enzymes. • Electrophoresis • Southern blotting • Hybridization with a radioactive probe • Process with X-ray film • In a three probe system of DNA typing, the typical DNA pattern will show a maximum of about 6 bands. • One band can indicate that the individuals are probably homozygous for the specific gene.

21. Gel Electrophoresis • DNA is separated by fragment size. • Smaller size fragments move faster than larger fragments.

22. Southern Blotting • The transfer of DNA fragments onto a nylon membrane.

23. Hybridization • Process of joining two complementary strands of DNA to form a double stranded molecule.

24. Gene Splicing • Laboratory procedure responsible for making possible the development of bacteria that can synthesize insulin.

25. Advantages of Working With Short DNA Fragments • They are more stable & less likely to break apart. • They are less subject to degradation due to adverse environmental conditions. • Quantity can be greatly amplified by PCR technology.

26. STR • Short Tandem Repeats • Region of a DNA molecule that contains short segments consisting of 3 -7 repeating base pairs. • Most successful & widely used DNA profiling process. • Commercial STR kits include means to detect the amelogenin gene to allow for gender determination.

27. STR vs RFLP • STR analysis has replaced RFLP DNA typing because: • It is less subject to sample degradation. • It reduces the time to obtain results from a sample. • It requires a smaller sample size.

28. STR Capillary Electrophoresis • Evolved from flat gel electrophoresis. • Automates sampling and data collection. • Decreases analysis time.

29. Multiplexing • The concept of simultaneously extracting, amplifying, and detecting a combination of STRs. • Y-STR markers are useful in cases where multiple males are involved in a sexual assault on a victim. (expect 3 peaks).

30. mtDNA • Mitochondrial DNA. • HV1 & HV2 are regions of mtDNA. • Few forensic labs do mtDNA analysis because • Analysis procedure is very rigorous. • Costs much more than nuclear DNA profiling. • Study takes time. • Discriminating power of mtDNA is less than that of STR analysis.

31. DNA-profiling technology • Has been used to • Settle matters of questioned paternity/maternity. • Match suspect(s) to biological evidence found at a crime scene. • Decide immigration cases based on family relationships.

32. CODIS • Shared databases of DNA typing information from convicted felons and crime scene evidence.

33. Luminol • Detects traces of blood without compromising potential DNA typing.

34. Buccal Cells • Cells obtained from the mouth and inside of the cheek.