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FORENSIC SCIENCE DNA

FORENSIC SCIENCE DNA. DNA testing creates patterns unique to each individual. Key Idea:. What is DNA? How does DNA make me unique? How is DNA used in profiling? Why is “junk” DNA used in forensic profiling?. Goals:. Historical Information .

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FORENSIC SCIENCE DNA

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  1. FORENSIC SCIENCE DNA

  2. DNA testing creates patterns unique to each individual. Key Idea:

  3. What is DNA? How does DNA make me unique? How is DNA used in profiling? Why is “junk” DNA used in forensic profiling? Goals:

  4. Historical Information • 1953--Watson and Crick discovered the structure of the DNA molecule • 1980--Ray White described the 1st polymorphic RFLP marker • 1985--Alec Jeffreys isolated DNA markers and called them DNA “fingerprints • 1985--Kary Mullis developed PCR testing • 1988--FBI starts DNA casework • 1991--First STR paper • 1998--FBI launches CODIS database • 2003--Completion of the Human Genome Project

  5. DNA Structure • Double helix--two coiled DNA strands • Nucleotides--each unit contains a sugar (deoxyribose), a phosphate group and a nitrogen-containing base • Four Bases • Adenine • Cytosine • Guanine • Thymine • Bases always pair A--T and C--G

  6. DNA Locations in the body • Can be found in all body cells--blood, semen, saliva, urine, hair, teeth, bone, tissue • Most abundant in our buccal (cheek) cells • Blood is 99.9% red blood cells that have no nuclei; and therefore, no nuclear DNA • DNA obtained from blood comes from white blood cells

  7. Non-Coding Regions • Only one-tenth of a single percent of DNA (about 3 million bases) differs from one person to the next. Scientists use these regions to generate a DNA profile of an individual. • 97% is non-coding and is repetitive • 3% of the human DNA sequences code for proteins • 50% of the human genome has these interspersed repetitive sequences

  8. DNA TYPING METHODS DNA typing methods create patterns that ultimately distinguish each individual.

  9. KEY TO UNDERSTANDING • The key to understanding DNA typing lies in the knowledge that within the world’s population numerous possibilities exist for the number of times a particular sequence of base letters can repeat itself on a DNA strand. Saferstein, Richard. Criminalistics An Introduction to Forensic Science

  10. Forensic Testing will take advantage of the repeating sections of DNA • Looks at the tandem repeats • There are many versions of some of these repeats (polymorphic) • Remember: Half of the DNA is from the mother, half from the father

  11. DNA TYPING“Fingerprinting”Methods • RFLP-- Restriction Fragment Length Polymorphism • PCR-- Polymerase Chain Reaction • STR--Short Tandem Repeats • Mitochondrial--use of maternal DNA in the mitochondria • y-STR-only found in males

  12. Restriction Fragment Length Polymorphism • Using restriction enzymes, differing lengths of chromosome fragments are separated by electrophoresis. • A DNA fragment pattern will be created by electrophoresis

  13. RFLP-Restriction Fragment Length Polymorphisms • Extract the DNA--from biological sample (blood, semen, saliva) • Add restriction enzymes--which will cut the DNA at specific regions • Use electrophoresis--to separate the DNA fragments by size • Detect regions--using luminescent dyes or radioactive probes that will expose X-ray film

  14. RFLP-Restriction Fragment Length Polymorphisms • Takes at least 10 weeks • Is very specific if multiple restriction enzymes are used • Requires a substantial sample size • The large number of base units (1000’s) per fragment make the fragments susceptible to breaking

  15. PCR--Polymerase Chain Reaction PCR is a technique for making many copies of a defined segment of a DNA molecule. It looks at several regions of repeated bases. Every gene has at least two alternative forms called alleles. An individual receives one allele from mother and one from father. If the alleles are the same, the individual is said to be homozygous for the trait; if the two alleles are different, the individual is heterozygous.

  16. PCR--Polymerase Chain Reaction steps • Heat the DNA strands to 94 degrees which causes the strands to separate (unzip). • Add primers to the separated strands. • The primers combine with the DNA strands. • Add DNA polymerase and a mixture of free nucleotides to the separated strands. • The tube is cooled down to 72 degrees which results in the rebuilding of a double-strand of DNA.

  17. PCR The outcome is a doubling of the number DNA strands. Repeat 25 to 30 times, yielding more than one million copies of the original DNA molecule. Each cycle takes less than two minutes from start to finish.

  18. Advantages of PCR • Minute amounts of DNA template may be used from as little as a single cell. • DNA degraded to fragments only a few hundred base pairs in length can serve as effective templates for amplification. • Large numbers of copies of specific DNA sequences can be amplified simultaneously with multiplex PCR reactions. • Commercial kits are now available for easy PCR reaction setup and amplification. Problem: Contaminant DNA, such as fungal and bacterial sources, will not amplify because human-specific primers are used.

  19. Short Tandem Repeats (STR) • STR typing uses the PCR method • STR’s are locations (loci) on the chromosome that contain short sequences of 3 to 7 bases that repeat themselves with the DNA molecule. • Advantages include a higher discrimination than RFLP, less time, smaller sample size, and less susceptible to degradation. • In Forensic Science, 13 locations are used.

  20. CODIS: Combined DNA Index System

  21. Example: Using STR • Extract the gene TH01 from the sample. (TH01 has seven human variants and a repeating sequence of A-A-T-G) • Amplify the sample by means of PCR • Separate by electrophoresis • Examine the distance the STR migrates to determine the number of times TH01 repeats

  22. Example con’t • Each person has two STR types for TH01--one inherited from each parent. • Continue the process with additional STRs from other genes Imagine how the probability of a match DECREASES by using an INCREASING number of STRs.

  23. STR Analysis • 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.

  24. Profiler Plus Allelic Ladders VWA D3S1358 FGA AMEL D8S1179 D21S11 D18S51 D13S317 D5S818 D7S820

  25. COfiler Allelic Ladders D3S1358 D16S539 AMEL TH01 TPOX CSF1PO D7S820

  26. My STR’s

  27. Probability of Matching My STR’s

  28. DNA Interactive Go to the website below to see a STR animation demonstration. Click on human identification , profiling and then on the third circle called Today’s DNA Profiling http://www.dnai.org/d/index.html

  29. Electrophoresis • An electrical current moves through a substance causing molecules to sort by size. • Smaller, lighter molecules will move the furthest on the gel.

  30. Three Possible Outcomes • Match--The DNA profile appears the same. Lab will determine the frequency. • Exclusion – The genotype comparison shows profile differences that can only be explained by the two samples originating from different sources. • Inconclusive - The data does not support a conclusion as to whether the profiles match.

  31. Fill with Polymer Solution Argon Ion Laser - + Inlet (cathode) Outlet (anode) 5-20 kV Capillary Electrophoresis (CE) 50-100 m x 27 cm Burn capillary window DNA Separation occurs in minutes... Data Acquisition and Analysis

  32. Gel Electrophoresis Capillary Electrophoresis capillaries laser gel plate

  33. DNA Analyst at work Computer Screen Data from capillary electrophoresis

  34. A comparison of the computer screen and a possible print out that represents the fragments by peaks.

  35. Electrophoresis Pipette the DNA. We are working with a smaller budget and less expensive equipment. This is what we will do in class.

  36. Electrophoresis (cont.) Load DNA into the gel wells.

  37. Electrophoresis (cont.) • Run the gel. • Observe and compare bands of DNA.

  38. Nuclear DNA Nuclear found in the nucleus Constitutes 23 pair of chromsomes inherited from both parents Each cell contains only one nuclei Mitochondrial DNA Found in the cytoplasm Is inherited only from mother Each cell contains hundreds to thousands of mitochondria Can be found in skeletal remains Types of DNA Nuclear DNA is present in the head of the sperm. Mitochondrial DNA is present in the tail. At conception, the head of the sperm enters the egg and unites with the nucleus. The tail falls off, losing the father’s mitochondrial DNA.

  39. Mitochondrial DNA • 37 genes are involved in mitochondrial energy generation • Is best used when nuclear DNA typing is not possible • Analysis of mtDNA is more: • Rigorous • Time consuming • Costly than nucleic testing of DNA

  40. Comparison of DNA Methods

  41. Determining Probability Databases are established by determining how often a particular allele on a loci appears in a given population. By increasing the number of alleles on different loci, the probability of having two people with the exact combination becomes astronomical.

  42. Probability of a Random Match Using 13 CODIS STR Markers

  43. Present Work in DNA • CODIS--Combined DNA Information System. • A data base of DNA profiles of individuals convicted of sex crimes and other violent crime • TWGDAM--The Working Group for DNA Analytical Methods • Wrote the standards for DNA analysis that are part of a national crime laboratory accreditation program • Innocence Project • Group of college law students work on cases where DNA may prove innocence (or guilt)

  44. FBI’s CODIS DNA Database Combined DNA Index System • Used for linking serial crimes and unsolved cases with repeat offenders • Launched October 1998 • Links all 50 states • Requires >4 RFLP • markers and/or • 13 core STR markers

  45. The Future • Greater automation of the DNA typing process • Use of SNP’s--single nucleotide polymorphism which measures a one nucleotide change or difference from one individual to another. More sites are needed to differentiate between individuals (30 to 50 SNPs to attain the frequencies of the 13 STR loci), but it can be done with robots and automation.

  46. DNA--The Search for Innocence • DNA implicates the guilty and exonerates the innocent--R. Saferstein Over the years many innocent people have been wrongly convicted with no way of proving their innocence. Since the late 1980’s, DNA technology has been available. People, as a result, have been reopening cases and testing the evidence for DNA. Some have been exonerated because their DNA did not match. In this unit you will research a case in which DNA was used to convict or vindicate the suspect. You will work with a partner and will present your findings to the class using PowerPoint. • http://www.dnai.org/d/index.html

  47. WORDS OF WISDOM Keep in mind a famous quote from Albert Einstein, “ If we knew what we were doing, it would not be called research, would it?”

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