Analysis in Forensic Science

Transcription

1 Chapter 16 Gene Cloning & DNA Analysis in Forensic Science 1. DNA analysis in identification of crime suspects 2. Studying kinship by DNA profiling 3. Sex identification by DNA analysis Forensic science is the final area of biotechnology that we will consider In popular media, these techniques are called genetic fingerprinting, though the more accurate term for procedures used today is DNA profiling 1

2 16.1 DNA analysis in the identification of crime suspects Genetic fingerprinting by hybridization probing DNA profiling by PCR of short tandem repeats DNA analysis in the identification of crime suspects It is probably impossible for a person to commit a crime without leaving behind a trace of his or her DNA Hairs, spots of blood & even conventional fingerprints contain traces of DNA Enough to be studied by polymerase chain reaction (PCR) 2

3 DNA analysis in the identification of crime suspects Identical twins are the only individuals have identical copies of human genome Human genome contains many polymorphisms nucleotide sequence is not the same in every member of population same as DNA markers in genome mapping Restriction fragment length polymorphisms (RFLPs), short tandem repeats (STRs) & single nucleotide polymorphisms (SNPs) Genetic fingerprinting by hybridization probing First method for using DNA analysis to identify individuals mid-1980s by Sir Alec Jeffreys of Leicester University Not based on polymorphic site, but on a different kind of variation in the human genome called a hypervariable dispersed repetitive sequence This is a repeated sequence that occurs at various places Their genomic positions are variable: they are located at different positions in genomes of different people 3

4 Figure 16.1 (a) Polymorphic repeat sequences in human genome The positions of polymorphic repeats, such as hypervariable dispersed repetitive sequences, in the genomes of two individuals In second person has an additional repeat sequence The particular repeat that was initially used in genetic fingerprinting contains the sequence GGGCAGGANG (where N is any nucleotide) Genetic fingerprinting by hybridization probing Figure 16.1(b) procedure A sample of DNA is digested with a restriction endonuclease & fragments separated by agarose gel electrophoresis & a Southern blot prepared Hybridization to blot of a labelled probe containing repeat sequence reveals a series of bands The same procedure carried out with a DNA sample from a second person will give a different pattern of bands 4

5 Figure 16.1 (b) Two genomic fingerprints An autoradiograph showing the genomic fingerprints of two individuals Strictly speaking, genetic fingerprinting refers only to hybridization analysis of dispersed repeat sequences Genetic fingerprinting by hybridization probing This technique has been valuable in forensic work but suffers from three limitations: (1) A relatively large amount of DNA is needed Fingerprinting cannot be used with the minute amounts of DNA in hair & bloodstains (2) Interpretation of fingerprint can be difficult In a court of law, minor differences in band intensity between a test fingerprint & that of a suspect can be sufficient for the suspect to be acquitted (3) Although insertion sites of repeat sequences are hypervariable, there is a limit to this variability Therefore a small chance that two unrelated individuals could have the same, or at least very similar, fingerprints The more powerful technique of DNA profiling avoids these problems Profiling makes use of the polymorphic sequences called STRs 5

6 DNA profiling by PCR of short tandem repeats (STRs( STRs) An STR A short sequence, one to 13 nucleotides in length, that is repeated several times in a tandem array In human genome, most common type of STR is dinucleotide repeat [CA] n where n, the number of repeats, is usually between 5 and 20 (Figure 16.2(a)) Number of repeats in a particular STR is variable because repeats can be added or, less frequently, removed by errors that occur during DNA replication In population as a whole, there might be as many as ten different versions of a particular STR, each of alleles characterized by a different number of repeats DNA profiling by PCR of short tandem repeats (STRs( STRs) Can be achieved quickly & with very small amounts of DNA by PCRs primers anneal to DNA sequences either side of a repeat (Figure 12.11) 6

7 DNA profiling by PCR of short tandem repeats (STRs( STRs) After the PCR products are examined by agarose gel electrophoresis size of band or bands indicating the allele or alleles present in the DNA sample Two alleles of an STR can be present in a single DNA sample because there are two copies of each STR one on chromosome inherited from mother & one on chromosome from father DNA profiling by PCR of short tandem repeats (STRs( STRs) Because PCR is used DNA profiling is very sensitive & enables results to be obtained with hairs & other specimens that contain just trace amounts of DNA The results are unambiguous, & a match between DNA profiles is usually accepted as evidence in a trial DNA profiling gives a very high statistical probability that a match between a test profile & that of a suspect is significant & not due to a chance similarity between two different people 7

8 DNA profiling by PCR of short tandem repeats (STRs( STRs) Necessary degree of certainty can be achieved by analysis of a panel of nine STRs can be typed in a single multiplex PCR, in which a series of primer pairs are used in a single reaction because the PCRs are designed so that the products obtained from each STR have different sizes so appear at different positions on agarose gel DNA profiling by PCR of short tandem repeats (STRs( STRs) Alternatively, if primers are labelled with different fluorochromes, results can be visualized by running products in an automated DNA sequencer (Figure 16.2(c)) (c) Analysis of multiplex PCR results in an automated DNA sequencer 8

9 16.2 Studying kinship by DNA profiling Kinship analysis: DNA profiling can also be used to infer if two or more individuals are members of the same family Its main day-to-day application is in paternity testing 16.2 Studying kinship by DNA profiling Related individuals have similar DNA profiles DNA profiling and the remains of the Romanovs 9

10 Related individuals have similar DNA profiles Relationships within a family therefore become apparent when the alleles of a particular STR are marked on the family pedigree (Figure 16.3) Figure 16.3 Inheritance of STR alleles within a family Related individuals have similar DNA profiles Three of four children have inherited the 12- repeat allele from the father This observation in itself is not sufficient to deduce that these three children are siblings To increase the degree of certainty, more STRs would have to be typed But the analysis need not be endless because a comparison of nine STRs gives an acceptable probability that relationships that are observed are real 10

11 DNA profiling and the remains of the Romanovs An interesting example During 1990s, on bones of the Romanovs, last members of the Russian ruling family Tsar Nicholas II was deposed at the time of Russian Revolution; he & his wife (Tsarina Alexandra) & their five children were imprisoned In 1917, all seven were killed along with their doctor & various servants In 1991, after the fall of communism, the bodies were recovered from their roadside grave The Romanov family tree The only juvenile bones among the collection should have belonged to the children of Tsar & Tsarina 11

12 The results of STR analysis THO1 data: Female adult 2 cannot be the mother Because she only possesses allele 6 None of the child have Exclude male adult 4 as a possible father But do not allow the other three male adults to be distinguished VWA/31 results Exclude male adults 1 & 2 But were these indeed the bones of the Romanovs or could they be the remains some other unfortunate group of people? Compared with DNA samples from living relatives of the Romanovs This work included studies of mitochondrial DNA, the small 16 kb circles of DNA contained in the energygenerating mitochondria of cells These mitochondrial DNA studies showed that the bones were indeed those of Tsar Nicholas, Tsarina Alexandra, & three of their daughters 12

13 The missing children Only three children were found in the Romanovs' grave Alexei, the only boy, & one of the four girls were missing During the middle decades of the twentieth century several women claimed to be a Romanov princess Regrettably, DNA testing has shown that none of these claimants could have been a daughter of the Tsar & Tsarina The story of Anastasia is probably just a romance 16.3 Sex identification by DNA analysis Its application to identify the sex of an individual Forensic scientists occasionally have to deal with bodies that are so badly damaged that DNA analysis is the only way of determining sex to identify the sex of an unborn child An example is when the pedigree of the family indicates that an unborn male might suffer from an inherited disease and the parents wish to make an early decision about whether to continue the pregnancy in the analysis of archaeological specimens If ancient DNA is preserved in the bones, a DNA-based method can tell the archaeologists if they are dealing with a male or a female 13

14 16.3 Sex identification by DNA analysis PCRs directed at Y chromosome-specific sequences PCR of the amelogenin gene PCRs directed at Y chromosome-specific sequences There are several repeated sequences that are only located in the Y chromosome These repeated sequences acting as multiple targets for the PCR & hence giving greater sensitivity A PCR directed at Y-specific DNA sequences would give a product with male DNA but no band if the sample comes from a female 14

15 Figure 16.5 Sex identification by PCR of a Y-specific DNA sequence Male DNA gives a PCR pruct (lane 2), but female DNA does not (lane 3) The problem is that a failed PCR (lane 4) gives the same result as female DNA This is a clear distinction between the two alternatives & hence a perfectly satisfactory system for most applications PCRs directed at Y chromosome- specific sequences if the sample did not contain any DNA if the DNA was too degraded to work in the PCR if the sample also contained inhibitors of Taq polymerase so that the PCR did not work? All of these possibilities could occur with archaeological specimens especially those that have been buried in the ground & become contaminated with humic acids & other compounds known to inhibit many of enzymes used in molecular biology research 15

16 PCR of the amelogenin gene The amelogenin gene codes for a protein found in tooth enamel It is one of the few genes that are present on Y chromosome &, like many of these genes, there is also a copy on X chromosome Figure 16.6 (a) An Indel in the amelogenin gene But the two copies are far from identical when the nucleotide sequences are aligned a number of indels are seen Indel: positions where a segment of DNA has either been inserted into one sequence or deleted from the other sequence 16

17 Figure 16.6 (b) The results of PCRs spanning the idel Male DNA gives two products: 106 & 112 bp Female DNA gives just the smallest product A failed PCR gives no products & so is clearly distinguishable from the two types of positive results The development of the amelogenin system for sex identification is having an important impact in archaeology 17