Forensic Science: Forensic Science: DNA Typing. The Basics

Transcription

1 Forensic Science: Forensic Science: The Basics DNA Typing Chapter 14

2 DNA 1980s forensic biology turned to DNA DNA enhanced potential for matching suspect to crime scene DNA can come from almost any biological evidence DNA has even been found on mummies

3 DNA Deoxyribonucleic acid (DNA) is a large polymeric molecule found in virtually every cell in the body Exceptions are red blood cells and nerve cells DNA is found in two regions of cell Nucleus and Mitochondria

4 Structure of DNA Structure of DNA

5 The Cell The Cell The smallest unit of life The nucleus is the brain of the cell contains all the genetic contains all the genetic info the cell needs to exist & to reproduce In most types of cells, genetic information is organized into structures called chromosomes

6 Chromosomes In most types of cells, genetic information is organized into structures called chromosomes usually X shaped Y Y chromosome in males 23 pairs in humans one from mother & one one from mother & one from father

7 Genes Each chromosome contains hundreds to thousands information blocks called genes Each gene is the blueprint for a specific type of protein in the body only identical twins will have all the genes identical

8 Chromosomes Each chromosome is a single polymeric molecule called DNA if fully extended the molecule would be about 1.7 meters long unwrapping all the DNA in all your cells cover the distance from earth to moon 6,000 times

9 Nuclear DNA Nuclear DNA Unique type of molecule Shape is double helix (consider a ladder) Poles of DNA are same in all people Rungs are made of bases or nucleotides There are four bases that make up these. Adenine (A) Thymine (T) Guanine (G) Cytosine (C)

10 DNA Only certain pairs of bases can join together No base can join itself A A strand of DNA has millions of base pairs and rules can not be violated

11 Cellular DNA Cellular DNA Characteristics are defined within genetic code that is contained within portions of chromosomes called genes Location of where a gene is found on a chromosome is called a locus Human genome contains more than 100,000 genes A gene that exists in more than one form is referred to as polymorphic Different forms are called alleles

12 Cellular DNA Cellular DNA Homozygous individual inherits same allele for a particular characteristic from both parents Heterozygous-individual inherits different allele with respect to gene Genes can be dominant or recessive

13 Nucleotides DNA is a polymer built from monomers called nucleotides Each nucleotide is consists of deoxyribose pentose sugar phosphoric acid a nitrogenous base

14 The phosphate The sugar

15 Nitrogenous Bases Nitrogenous Bases The Purines Adenine (A) Guanine (G)

16 Nitrogenous Bases Nitrogenous Bases The Pyrimidines Cytosine (C) Thymine (T)

17 The DNA Backbone The DNA Backbone The monomers are linked together by phosphodiester bridges (bonds) links the 3 carbon in the ribose of one nucleotide to the 5 carbon in the ribose of the adjacent nucleotide

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19 The DNA Double Helix The DNA Double Helix DNA is normally a double stranded macromolecule Two polynucleotide chains are held together by H-bonding together by H-bonding A always pairs with T C always pairs with G

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22 5 T-T-G-A-C-T-A-T-C-C-A-G-A-T-C 3 3 A-A-C-T-G-A-T-A-G-G-T-C-T-A-G 5 In a double helix the strands go in opposite directions

23 Functions of DNA Functions of DNA Two Functions To transmit information from one generation of cells to the next To provide the information for the synthesis of components (proteins) necessary for cellular function

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25 DNA Fingerprinting DNA Fingerprinting The basic structure of everyone s DNA is the same the difference between people is the ordering of the difference between people is the ordering of the base pairs Every person can be distinguished by the Every person can be distinguished by the sequence of their base pairs millions of base pairs make this impractical a shorter method uses repeating patterns that are present in DNA

26 VNTR s DNA strands contain information which directs an organism s development exons Also contain stretches which appear to provide no relevant genetic information introns repeated sequences of base pairs Variable Number Tandem Repeats (VNTRs) can contain anywhere from 20 to 200 base pairs

27 VNTRs All humans have some VNTRs VNTRs come from the genetic information VNTRs come from the genetic information donated by parents can have VNTRs from mother, father or a can have VNTRs from mother, father or a combination will not have a VNTR that is from neither parent

28 D1 = biological daughter of both parents D2 = child of mother & former husband S1 = couple s biological son S2 = adopted son

29 VNTR Analysis VNTR Analysis Usually an individual will inherit a different variant of the repeated sequence from each parent

30 VNTR Analysis VNTR Analysis PCR primers bracket the locus PCR reaction forms a nucleotide chain from the template

31 VNTR Analysis VNTR Analysis The length of the amplified DNA & its position after electrophoresis will depend on the number or repeated bases in the sequence

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33 Analysis used 3 different VNTR loci for each suspect giving 6 bands

34 Although some individuals have several bands in common, the overall pattern is distinctive for each

35 Suspects A & C can be eliminated B remains a suspect

36 DNA Is better for identification than polymorphic substances due to: Many polymorphic enzymes and antigens do not survive Is better for identification than polymorphic Many polymorphic enzymes and antigens do not survive drying process Even if many are measured in an individual person, there is not enough total variation from one person to another to use groupings to definitively associate a person with biological evidence Polymorphic substances are mainly in blood, not other biological tissue

37 Two Types of Polymorphism Two Types of Polymorphism Sequence polymorphism- occurs when there is a difference in one or more base pairs within a gene Length polymorphism- occurs in strands of DNA where repeating sequences of base pairs are encountered Repeats occurring next to each other without any intervening base pairs are referred to as tandem repeats

38 Interpreting DNA Evidence: Interpreting DNA Evidence: Population Genetics Forensic scientists compares DNA from biologic evidence from crime scene with known DNA from suspect or victim Focus on length or sequence polymorphisms because those are parts that differentiate people More than 99% of every person s DNA is identical Differences in DNA from one person to another is less than.1% of a person s DNA

39 Interpreting DNA Evidence: Interpreting DNA Evidence: Population Genetics The more parts of DNA a forensic scientists examine, the more certain the scientists can be that the DNA came from a particular person Product rule- states the probability of two or more independent events occurring is the product of probabilities of each event

40 Probability Forensic scientists use probability to interpret likelihood DNA came from a particular person At each locus where DNA type is to be analyzed, scientists have determined population frequency of that allele Product rule can be used to determine overall probability of having all of the alleles

41 Collection and Preservation of DNA Collection and Preservation of DNA Evidence DNA can be found in may places- licked stamps, toothbrushes, pillows, inside a hat, discarded chewing gum Special care must be taken in collecting DNA Always assume it is infectious Protective clothing should be worn to minimize risk of infection, and make sure collector does not contaminate DNA sample

42 Collection and Preservation of DNA Collection and Preservation of DNA Evidence Elimination samples are known samples of DNA collected from all personnel at scene who could have contributed DNA Biologic evidence never should be packaged in airtight containers (bacteria build up from moisture can degrade DNA) Paper bags or breathable containers should be used Substrate control- sample that does not contain biologic material and serves as a control

43 Collection and Preservation of DNA Collection and Preservation of DNA Evidence Known samples of DNA are usually collected from gently scraping inside of the cheek- known as a buccal sample In the event of blood samples, place in test tube with preservative such as EDTA (ethylenediamine tetraacetic acid)

44 DNA Typing DNA Typing Parts of DNA are identified that are polymorphic and all of the alleles are known and their population frequencies are measured Ways of DNA typing are; RFLP PCR STR

45 Restriction Fragment Length Restriction Fragment Length Polymorphism (RFLP) First commercial technique for DNA typing Genes that are coded for desirable trait is spliced out of DNA by restriction enzymes or endonucleases RFLP uses restriction enzymes to cut DNA at certain polymorphic regions Hypervariable regions contain a large number of alleles

46 RFLP Traditionally accomplished using gel electrophoresis Results in DNA strands becoming denatured, or the double helix breaking apart where base pairs connect with each other After separation, they are transferred to a nylon membrane using a technique known as Southern Blotting

47 RFLP Repeating fragments are labeled next by adding probe to the DNA on the nylon membrane Process of adding a labeled probe is known as probe hybridization DNA ladders are lanes that that have many strands of known length DNA for calibration purposes

48 RFLP Problems RFLP Problems Repeating DNA strands are quite long, often with thousands of base pairs When DNA degrades, long strands tend to cleave in unpredictable location If degradation is advanced enough, may not be sufficient DNA to type by RFLP RFLP requires large amount of material If small quantity (few micrograms of DNA) is present, may not be sufficient to type

49 Polymerase Chain Reaction (PCR) Polymerase Chain Reaction (PCR) 1983 Dr. Kary Mullis developed reliable method of cloning or amplifying DNA Can be used to clone part of a strand of DNA First method adapted for forensic use as a means of amplifying DNA so that a sufficient quantity would be available for RFLP

50 DNA Amplification DNA Amplification Uses DNA polymerase, an enzyme present in all living organisms During cell division DNA denatures, becoming single stranded DNA polymerase catalyzes the addition of complimentary base pairs to the DNA, forming double helix strands Mullis developed Thermal Cycler, in which the PCR process takes place

51 DNA Amplification DNA Amplification Takes place in three steps; Denaturation mixture heated close to boiling Denaturation mixture heated close to boiling causing strands of DNA to denature Annealing temperature lowered and primers added to start formation of new double stranded DNA Extension- in presence of DNA polymerase as a catalyst, base adds to open position next to primer according to the base that is already on the single strand- New double strands complete

52 How PCR Amplified DNA is Typed How PCR Amplified DNA is Typed Certain polymorphic loci were chosen that could be amplified then typed in one operation HLA DQα human leukocyte antigen that exhibit sequence polymorphism Method for determining which allele is present is called reverse dot blot Amplified DNA placed on specially treated nylon strips that contain alleles and color forming reagents- at matches, colors change

53 PCR Typing Problems PCR Typing Problems Genes and other sites that are best are not very polymorphic and even the rarest types have high population frequencies PCR cannot distinguish separate DNA types in mixtures

54 Short Tandem Repeats (STRs) Short Tandem Repeats (STRs) Developed in middle 1990s. A method of DNA typing developed that combined strengths of PCR and RFLP while minimizing disadvantages STR are loci on chromosomes that repeat like those used in RFLP, but repeating sequence is much shorter, currently 3-7 base pairs long Entire STRs are a few hundred pairs long, compared to thousands of RFLP

55 STR STR analysis starts with PCR Locus of interest is identified and amplified by PCR Amplified fragments are separated and displayed using capillary electrophoresis 13 STR loci are currently amplified and analyzed in forensic science cases See table 14.1 Population Statistics for 13 Loci

56 Determination of Gender Determination of Gender Two approaches to gender determination Analysis of locus called amelogenin Females show one band of amelogenin while males show two upon analysis Analyze STRs that are present only on the Y chromosome- Y-STRs

57 Mitochondrial DNA Mitochondrial DNA At times, DNA is so degraded, nuclear DNA is not available so no analysis can take place (only have skeletal remains) Mitochondrial DNA is found in 1% of body Mitochondria are responsible for energy production in every cell of body Thousands of copies of mitochondrial DNA available in cell while only few copies of Nuclear DNA

58 Mitochondrial DNA Mitochondrial DNA Differs from nuclear DNA in important ways Arranged circularly, not in double helix 37 genes in mitochondrial DNA that direct energy production, but forensically important part consists of 1100 base pairs within two regions that do not have a genetic code function Regions are hypervariable- named HV1 and HV2

59 Mitochondrial DNA Mitochondrial DNA Mitochondrial DNA are only inherited from mother- no contribution from father Mitochondrial DNA typing is a useful vehicle for tracing one s parentage maternally While mitochondrial DNA exhibits a great deal of variability among unrelated people, there are only 2 regions that exhibit this Mitochondrial DNA typing is not able to individualize DNA currently

60 CODIS- Combined DNA Index CODIS- Combined DNA Index System Is the development of local, state and national databases that contain DNA types of many people who have been involved in crime CODIS began in 1990 Contains three layers- local, state and federal levels Is held back by lack of funding to crime laboratories to process the samples

61 CODIS Active cases are the priority Old cases that had biologic evidence (cold cases) are now being processed All data in CODIS is entered by same type Thirteen loci are standard loci for CODIS Each sample must have a DNA type at those loci FBI reports 27,000 investigations have been aided by CODIS