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Dna fingerprinting

1. 1. DNA FINGERPRINTING
2. 2. What is DNA fingerprinting ? <ul><li>It is a method of identifying an individual by unique characteristics of that person’s DNA </li></ul>
3. 3. A Historical Perspective <ul><li>In the course of his research on variability in human DNA, Alec Jeffreys developed a method of forensic DNA typing. </li></ul><ul><li>This method, termed ‘ DNA Fingerprinting’ , was used for the first time to solve two rape/murder cases in the UK in 1987. </li></ul><ul><li>Jeffreys was knighted in 1994 for Services to Science, and has been the recipient of numerous other honours </li></ul>
4. 4. DNA Fingerprinting & DNA Profiling - same or different ? <ul><li>DNA fingerprinting , as developed by Sir Alec Jeffries, targeted particular repeating sequences of DNA (9-80 bp long) found at a number of loci (multilocus). Jeffries described the pattern produced in a “fingerprint” as unique to an individual. Technology at the time (1985) required good DNA samples and took 1 - 2 weeks for a result. </li></ul><ul><li>Advances in technology have led to DNA profiling , using smaller short tandem repeats ( STR s) also from a number of loci. The smaller STRs are more likely to survive DNA degradation, use less DNA (because of PCR technology), and can be processed within 24 hours. </li></ul>
5. 5. Stages of DNA Profiling <ul><li>Stage 1 : </li></ul><ul><li>Cells are broken down </li></ul><ul><li>to release DNA </li></ul><ul><li>If only a small amount of DNA is available it can be amplified using the polymerase chain reaction (PCR) </li></ul>
6. 6. Stages of DNA Profiling <ul><li>Step 2: </li></ul><ul><li>The DNA is cut into fragments using restriction enzymes . </li></ul><ul><li>Each restriction enzyme cuts DNA at a specific base sequence. </li></ul>
7. 7. Stages of DNA Profiling <ul><li>The sections of DNA that are cut out are called restriction fragments. </li></ul><ul><li>This yields thousands of restriction fragments of all different sizes because the base sequences being cut may be far apart (long fragment) or close together (short fragment). </li></ul>
8. 8. Stages of DNA Profiling <ul><li>Stage 3: </li></ul><ul><li>Fragments are separated on the basis of size using a process called gel electrophoresis. </li></ul><ul><li>DNA fragments are injected into wells and an electric current is applied along the gel. </li></ul>
9. 9. Stages of DNA Profiling <ul><li>DNA is negatively charged so it is attracted to the positive end of the gel. </li></ul><ul><li>The shorter DNA fragments move faster than the longer fragments. </li></ul><ul><li>DNA is separated on basis of size. </li></ul>
10. 10. Stages of DNA Profiling <ul><li>A radioactive material is added which combines with the DNA fragments to produce a fluorescent image. </li></ul><ul><li>A photographic copy of the DNA bands is obtained. </li></ul>
11. 11. Stages of DNA Profiling <ul><li>Stage 4: </li></ul><ul><li>The pattern of fragment distribution is then analysed. </li></ul>
12. 12. Uses of DNA Profiling <ul><li>DNA profiling is used to solve crimes and medical problems </li></ul>
13. 13. Crime <ul><li>Forensic science is the use of scientific knowledge in legal situations. </li></ul><ul><li>The DNA profile of each individual is highly specific. </li></ul><ul><li>The chances of two people having exactly the same DNA profile is 30,000 million to 1 (except for identical twins). </li></ul>
14. 14. Biological materials used for DNA profiling <ul><li>Blood </li></ul><ul><li>Hair </li></ul><ul><li>Saliva </li></ul><ul><li>Semen </li></ul><ul><li>Body tissue cells </li></ul><ul><li>DNA samples have been obtained from vaginal cells transferred to the outside of a condom during sexual intercourse. </li></ul>
15. 15. DNA Profiling can solve crimes <ul><li>The pattern of the DNA profile is then compared with those of the victim and the suspect. </li></ul><ul><li>If the profile matches the suspect it provides strong evidence that the suspect was present at the crime scene ( NB:it does not prove they committed the crime). </li></ul><ul><li>If the profile doesn’t match the suspect then that suspect may be eliminated from the enquiry. </li></ul>
16. 16. Example <ul><li>A violent murder occurred. </li></ul><ul><li>The forensics team retrieved a blood sample from the crime scene. </li></ul><ul><li>They prepared DNA profiles of the blood sample, the victim and a suspect as follows: </li></ul>
17. 17. Was the suspect at the crime scene? Suspects Profile Blood sample from crime scene Victims profile
18. 18. Solving Medical Problems <ul><li>DNA profiles can be used to determine whether a particular person is the parent of a child. </li></ul><ul><li>A childs paternity (father) and maternity(mother) can be determined. </li></ul><ul><li>This information can be used in </li></ul><ul><li>Paternity suits </li></ul><ul><li>Inheritance cases </li></ul><ul><li>Immigration cases </li></ul>
19. 19. An Example Using EcoR I for a Question of Paternity <ul><li>EcoR I cuts a similar section of DNA on Bob, Larry, and Mary </li></ul><ul><li>After the cut how many fragments Bob, Larry, and Mary have? </li></ul><ul><li>Answer: 2, 3, and none </li></ul>Recall: EcoR I cuts only at GAATTC
20. 20. Example Con’t <ul><li>After the DNA is cut with EcoR I, Bob’s, Larry’s and Mary’s fragments are placed in different lanes on an agarose gel </li></ul><ul><li>The fragments are then subjected to an electric field </li></ul><ul><li>The smaller fragments move faster, the larger ones move slower </li></ul><ul><li>This process of separating the fragments by length is called electrophoresis. </li></ul>
21. 21. Resulting Picture after Electrophoresis <ul><li>The bigger fragments are near the top </li></ul>
22. 22. Paternity Test <ul><li>In general the child’s DNA must be a combination of Mary’s DNA and one of the men. Which man is the father? </li></ul><ul><li>Answer: Larry </li></ul>
23. 23. What is Analyzed in the DNA ? <ul><li>DNA profiling depends on regions of non-coding DNA that show great variability between individuals (are polymorphic which means many forms) </li></ul><ul><li>Modern profiling uses Short Tandem Repeats , STRs </li></ul><ul><li>These are short sequences of DNA, usually 2-5 base pairs (bp) long, that repeat, or ‘stutter’ many times </li></ul>
24. 24. Short Tandem Repeats (STRs) the repeat region is variable between samples while the flanking regions where PCR primers bind are constant 7 repeats 8 repeats Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another AATG
25. 25. An Example of a STR in locus D7S280 <ul><li>D7S280 is a region ( locus ) of human chromosome 7. Its DNA sequence, as obtained from GenBank (a public DNA database) is: </li></ul><ul><li>1 aatttttgta ttttttttag agacggggtt tcaccatgtt ggtcaggctg actatggagt </li></ul><ul><li>61 tattttaagg ttaatatata taaagggtat gatagaacac ttgtcatagt ttagaacgaa </li></ul><ul><li>121 ctaac gatag atagatagat agatagatag atagatagat agatagatag atagacagat </li></ul><ul><li>181 tgata gtttt tttttatctc actaaatagt ctatagtaaa catttaatta ccaatatttg </li></ul><ul><li>241 gtgcaattct gtcaatgagg ataaatgtgg aatcgttata attcttaaga atatatattc </li></ul><ul><li>301 cctctgagtt tttgatacct cagattttaa ggcc </li></ul><ul><li>The STR repeat sequence is gata </li></ul><ul><li>Different alleles of this locus have from 6 to 15 tandem repeats of the ‘gata’ sequence </li></ul>
26. 26. New Technology <ul><li>STR analysis has largely replaced the original RFLP analysis (DNA Fingerprinting) developed in 1985 by Dr Alec Jeffreys </li></ul><ul><li>RFLP analysis requires good amounts of non-degraded DNA but STR analysis can be done on less than one billionth of a gram (a nanogram ) of DNA (as in a single flake of dandruff) </li></ul>
27. 27. DNA Fingerprinting & DNA Profiling - same or different ? <ul><li>DNA fingerprinting , as developed by Sir Alec Jeffries, targeted particular repeating sequences of DNA (9-80 bp long) found at a number of loci (multilocus). Jeffries described the pattern produced in a “fingerprint” as unique to an individual. Technology at the time (1985) required good DNA samples and took 1 - 2 weeks for a result. </li></ul><ul><li>Advances in technology have led to DNA profiling , using smaller short tandem repeats ( STR s) also from a number of loci. The smaller STRs are more likely to survive DNA degradation, use less DNA (because of PCR technology), and can be processed within 24 hours. </li></ul>
28. 28. Some uses of DNA Profiling <ul><li>Forensic work on crime scenes </li></ul><ul><li>Parentage testing (explored in more detail) </li></ul><ul><li>Victim identification in mass disasters </li></ul><ul><li>Animal identification- e.g. racehorses </li></ul><ul><li>Conservation biology and evolutionary studies </li></ul>
29. 29. Parentage Testing as conducted at DNA Diagnostics, Auckland
30. 30. Why Test ? <ul><li>Parentage - e.g. disputes over who is the father of a child & is thus responsible for child support </li></ul><ul><li>Determining whether twins are identical or fraternal </li></ul><ul><li>Estate cases (these may involve obtaining pathology samples of deceased individuals) </li></ul><ul><li>Immigration - establishing that individuals are the true children/parents/siblings in cases of family reunification </li></ul>
31. 31. Why Test? ctd <ul><li>Bone marrow transplant monitoring - to check that the transplanted marrow is still present </li></ul><ul><li>Determination of maternal cell contamination in chorionic villus sampling (used to investigate the possibility that a fetus has a severe inherited disease)- is the tissue sample really fetal? </li></ul><ul><li>Etc. </li></ul>
32. 32. The Steps <ul><li>Identification is established, by photo ID or by identification by a legal representative </li></ul><ul><li>A consent form is signed and witnessed </li></ul><ul><li>A case number is assigned </li></ul>
33. 33. The Steps, II <ul><li>DNA samples are collected- in the case of parentage testing, from the mother, child and putative (possible) father(s) </li></ul><ul><li>They are usually blood, but a buccal (cheek cell) swab is acceptable </li></ul>
34. 34. The Steps, III <ul><li>If the samples need transport they must be sent in leak proof containers for the courier’s safety. </li></ul>
35. 35. The Steps, IV <ul><li>The samples are processed, and DNA is extracted from each </li></ul><ul><li>Primers for each locus are added. Each primer is labeled with a fluorescent marker </li></ul>
36. 36. The Steps, IV, ctd <ul><li>DNA Diagnostics currently uses an AmpFlSTR Identifiler TM PCR Amplification Kit which targets 15 STR regions plus a sex specific region. </li></ul><ul><li>Kits allow standardization and accuracy, as DNA samples are added to a pre-made mix </li></ul>
37. 37. The Steps, V <ul><li>The DNA and fluorescent primers are run through the polymerase chain reaction (PCR) to amplify the targeted STR regions on the DNA </li></ul><ul><li>The samples are audited continually to ensure accuracy </li></ul>
38. 38. The Steps, VI <ul><li>The amplified DNA in a sample is separated by electrophoresis in a genetic analyzer </li></ul><ul><li>The analyzer has a gel-filled capillary tube through which the DNA travels (this replaces the gel slab of earlier days) </li></ul><ul><li>DNA fragments move through the gel tube by size, smallest first </li></ul><ul><li>A laser reads the fluorescent marked DNA loci </li></ul>
39. 39. An ABI Prism 310 Genetic Analyser Capillary tube Sample tray Note-other models of this Analyzer have more capillary tubes and can process more samples at a time, but this model is sufficient for the demand for testing to date through DNA Diagnostics
40. 40. Analyzing the Read-out <ul><li>Digital output from the Analyzer is read and interpreted by genotyping software </li></ul><ul><li>Each STR region read has two peaks, for the regions ( loci ) on an individual’s maternal and paternal chromosomes with that locus. note - if both regions are the same length, there is one peak </li></ul><ul><li>Data is shown both graphically and numerically </li></ul>Locus D19S433 = 14,15 Locus vWA = 15,16 Locus TPOX= 8,8 Locus D18S51= 13,16 A sample showing 4 loci- The top line is a ‘ladder’ for comparison
41. 41. A sample print -out for one person, showing all loci tested. Different colours help with interpretation
42. 42. Whose STR? <ul><li>A child will inherit one of the STRs at each locus from its mother, and since usually in parentage tests these are determined, then by elimination the other STRs at each locus come from its father </li></ul><ul><li>The father can donate either of his two STRs at each locus </li></ul><ul><li>If a child has STRs different from those of the putative father, then that man can be eliminated as a possible father </li></ul><ul><li>If a child has a particular STR that is the same as the putative father, it is necessary to examine possible matches with other STR loci and examine probability in Parentage Analysis </li></ul>
43. 43. Parentage Analysis <ul><li>For each STR tested, the data obtained is used to calculate a paternity index (the probability of the evidence given that a particular man is the father versus he is not the father) </li></ul><ul><li>This is based on the frequency in the population of the alleles at that locus </li></ul><ul><li>In New Zealand there are databases for European, Maori/Cook Islander, Asian and Tongan/Samoan. International databases are used for other ethnicities </li></ul>
44. 44. Analysis II <ul><li>Each STR site index is an independent event, so using probability law that says “ the probability that two independent events may happen together is the product of their individual probabilities ”, an overall paternity index is calculated by multiplying together the indices for each locus </li></ul>
45. 45. Parentage Analysis II, ctd Paternity index The index in this man’s analysis shows that the DNA evidence is 25 million times more likely that he is the biological father versus he is not (odds 25 million:1)
46. 46. Finally <ul><li>Further auditing and cross-checking is done to ensure accuracy </li></ul><ul><li>Parentage testing results in a report that is sent to all parties tested </li></ul>