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DNA Analysis Chapter 11 ©2010 Elsevier, Inc. INTRODUCTION • Serology has several drawbacks – Lack of stability of many proteins and limited ability to discriminate among a population of people • DNA has increased understanding of the development of plants, animals and diseases – Profound impact on forensic science ©2010 Elsevier, Inc. THE NATURE OF DNA • DNA (deoxyribonucleic acid) is a molecule found in nearly all cells – DNA is a polymer, a molecule made up of repeating simpler units, called monomers – Located in nucleus and mitochondria ©2010 Elsevier, Inc. Nuclear DNA – Structure is a double helix • A helix is a spiral-shaped object • Sugar molecules and phosphates connect the helices – Four base nucleotides: adenine (A), guanine (G), cytosine (C), and thymine (T) dangle off of each sugar molecule – A and T will bond with one another – C and G will bond with one another ©2010 Elsevier, Inc. Nuclear DNA – DNA molecule consists of sugar-phosphate backbones connected by linked base pairs: A-T, TA, G-C, C-G • Order of these pairs is controlled by genetic code, or blueprint that determines the characteristics of a person ©2010 Elsevier, Inc. DNA in Cells – Most cells have a nucleus, where most of cell functions are controlled – Within nucleus, DNA is arranged into 46 structures called chromosomes • Chromosomes are arranged into 23 pairs – One of each pair comes from father’s sperm and the other from the mother’s egg – One pair of chromosomes determines the sex of the individual ©2010 Elsevier, Inc. DNA in Cells ©2010 Elsevier, Inc. Genes and the Genetic Code • Genes are sections within the long strands of DNA – Ordering of base pairs in genes provides chemical instructions to manufacture particular proteins in the body – Genetic instructions are copied onto RNA (ribonucleic acid), which transmits this information to protein manufacturing sites within the cells • More than 99% of human DNA is exactly the same, less than 1% differentiates one human being from another ©2010 Elsevier, Inc. Variations of Genes: Alleles – Some traits are determined by a single gene on one chromosome; others are determined by multiple genes at several locations • If a person inherits the same form of a gene from the mother and the father, the person is said to be homozygous • If a person inherits different forms of the same gene, the person is said to be heterozygous • Different forms of the same gene or other DNA fragment at the same locus are called alleles • Some alleles are dominant and some are recessive – Variation is basis for DNA profile • The visible manifestation of variability of alleles is called phenotype • Genotype is the genetic description of the allele ©2010 Elsevier, Inc. Variations of Genes: Alleles – In DNA analysis, loci that are polymorphic are purposely chosen • More discriminating analysis – Two types of variability in alleles • Sequence polymorphism: Two sequences of double stranded DNA are exactly the same except at one location ©2010 Elsevier, Inc. Variations of Genes: Alleles • Length polymorphism: Consists of a series of base pairs that are repeated – Base pairs that repeat without any intervening pairs are referred to as tandem repeats – When variation in the number of repeats occurs from one individual to the next, the locus is described as having a variable number of tandem repeats (VNTR) – DNA type is a description of the types of alleles at all of the locations being analyzed on the genome ©2010 Elsevier, Inc. Population Genetics • Science of population genetics answers questions about DNA typing and likelihood of matching DNA – Frequency of occurrence for alleles analyzed are determined – Multiply the frequency of occurrence for each allele to determine the frequency of occurrence of all of these alleles at one time • Technique of multiplying probabilities together is called the product rule ©2010 Elsevier, Inc. Population Genetics • To determine an overall DNA type, each data point must be independent of the other data points – Loci used in genotyping have been tested to check for independence – Product rule has yielded genotypes that are so rare that the chances of reoccurrence within the population is extremely small ©2010 Elsevier, Inc. DNA TYPING • Restriction Fragment Length Polymorphism (RFLP) was the first DNA typing method to be widely used by forensic biologists • Polymerase chain reaction (PCR) is now used to increase the amount of DNA by amplification • Most laboratories use a typing method known as short tandem repeats (STR) ©2010 Elsevier, Inc. Restriction Fragment Length Polymorphism (RFLP) – DNA is extracted from biologic material and then severed into small fragments call minisatellites or variable number tandem repeats (VNTR) using restriction enzymes • Length polymorphism is used to discriminate a population of people. ©2010 Elsevier, Inc. How RFLP Works – Pieces of DNA are cut out of strand by restriction enzymes, also known as endonucleases • Designed to cut DNA at specific sequence of bases – In RFLP, polymorphic regions of DNA are identified • Regions are hypervariable; they have a large number of alleles ©2010 Elsevier, Inc. How RFLP Works – RFLP typing in the US predominantly used single locus VNTR analysis • A result of problems with limited or degraded DNA – Separation of DNA Fragments • Gel electrophoresis is used to separate fragments of DNA – Calibration standards, or ladders, are used to determined the lengths of the VNTRs when the gel is developed ©2010 Elsevier, Inc. How RFLP Works • A technique known as Southern Blotting is used to transfer the separated DNA fragments onto a more rugged nylon membrane ©2010 Elsevier, Inc. Visualization of VNTRs • Probe hybridization is used to visualize the VNTRs • Chemiluminescence or radio-labeled DNA are used for visualization – Exposed film is called an autorad • VNTR fragments cannot be viewed as discrete alleles, rather a band – Match probabilities can be calculated ©2010 Elsevier, Inc. The Polymerase Chain Reaction (PCR) • Polymerase chain reaction is used for making copies of DNA using polymerase enzymes – Overcame issue of relatively large quantities required for RFLP ©2010 Elsevier, Inc. The PCR Process – Sensitive to contamination; performed in location physically isolated from where subsequent amplifications will be performed – Three steps to process take place in thermal cycler, an apparatus capable of achieving and maintaining preset temperatures very precisely • Denaturation: The double stranded DNA denatures; bonds break between the base pairs that hold the strands together – Result is single stranded DNA – Strands are templates for formation of a new piece of double stranded DNA ©2010 Elsevier, Inc. The PCR Process • Annealing: Attach a short strand of synthetic DNA to each of the separated strands – Called primers because they will mark the starting points for addition of new bases to complete the reproduction of each strand • Extension: Single bases, or nucleotides, are added to the primer – Strand is built up and a new piece of double stranded DNA is produced – Strand is built up and a new piece of double stranded DNA is produced – Cycle of three steps is repeated until sufficient DNA is produced; 25-40 cycles ©2010 Elsevier, Inc. The PCR Process ©2010 Elsevier, Inc. DNA Typing of PCR Product – Very sensitive methods are not required, can run a yield gel experiment on agarose and stain with ethidium bromide – Human Leukocyte Antigen (HLA) DQ alpha gene was the first DNA region widely subjected to amplification and typing • DQ alpha and a number of other genes called polymarker are typed using a method called reverse dot blot – Involves identifying the particular alleles present by reacting them with color forming reagents on specially treated nylon strips – Not often used in forensic science » Not able to associate sample with just one individual » Not capable of resolving multiple DNA types in mixtures ©2010 Elsevier, Inc. Short Tandem Repeats (STRs) • STRs are length polymorphic sequences of short strands of DNA that range from two to six base pairs long and that repeat in tandem – Short tandem repeats are called microsatellites – Longer repeats, typed by RFLP, are called minisatellites • STR markers exhibit high variability in a population, are less sensitive to degradation of the DNA, many microsatellites to choose from • Combined DNA Index System (CODIS) is a standard database, containing 13 loci each with four base pair repeats – Gender is determined by analyzing the Amelogenin locus • DNA is amplified by PCR and separated ©2010 Elsevier, Inc. STRs • Capillary electrophoresis is used and DNA is detected by means of laser-induced fluorescence – Results in electropherogram – Large amount of data for analysis – Allelic ladders are strands of DNA made up of all common alleles present at each STR locus and are used for calibration ©2010 Elsevier, Inc. STRs • FBI decided that any DNA type whose odds of a chance occurrence exceeded 1,000 times the U.S. population would be considered individualized – All associations expressed as probabilities – Identical twins have the same DNA, at least insofar as forensic DNA methods are concerned – Care should be taken when evaluating such statistics of rare occurrence ©2010 Elsevier, Inc. Gender Identification – Two approaches to gender identification using DNA typing • Amelogenin is a locus on one of the chromosomes and can be used to determine sex • Second approach uses Y-STRs to determine gender – Useful when mixed samples are present ©2010 Elsevier, Inc. Mitochondrial DNA (mtDNA) • Mitochondria are small structures located within practically all animal and plant cells – Serve as energy mediators of the cell • Differences between mtDNA and genomic DNA ©2010 Elsevier, Inc. Mitochondrial DNA (mtDNA) • Number of differences between mtDNA and genomic DNA – mtDNA is circular in shape – Much less mtDNA is present; more copies of mtDNA in mitochondria cells as compared to few copies of genomic DNA – mtDNA contains non-coding region, which can be useful for comparing known and questioned DNA samples – All mtDNA comes from the mother – mtDNA shows a high degree of variation between unrelated people – mtDNA is useful in typing samples that have low quantities of DNA, are degraded or are very old • Most forensic laboratories that do genomic DNA typing also do mtDNA typing ©2010 Elsevier, Inc. INTERPRETATION OF DNA TYPING RESULTS • Purity Issues – Difficulty in interpretation comes from DNA that has been compromised in some way • Contamination: Introduction of foreign material • DNA from more than one source • Degradation: Break down due to extreme heat, light, time, humidity, biological causes and presence of certain chemicals • Extraneous materials: I.e. clothing dyes, blood components, soil ©2010 Elsevier, Inc. Comparison of DNA Samples • Rigorous scientific testing has been done that validates the concepts and testing methodologies for human DNA typing • Valid scientifically determined statistical frequencies for the alleles at the loci that are used in DNA typing have been calculated – Statistical methods are used – Term commonly used to express this comparison is “match” • No significant or unexplainable differences between the known and unknown materials – Rather than “match” the term “genetic concordance” is used to describe the relationship between two DNA samples ©2010 Elsevier, Inc. Estimation of Population Frequencies – Large numbers of people are tested to determine population frequency • Number of times each allele appears in population – Determining population frequency for an entire DNA profile is not so simple • Population frequencies of genotypes made up of multiple loci can be calculated by taking the product of the population frequencies of each individual locus ©2010 Elsevier, Inc. Interpretation of DNA Typing Results: Purity Issues • Contamination – Difficulty in interpretation comes from DNA that has been compromised in some way • Contamination: Introduction of foreign material • DNA from more than one source • Degradation – Degradation: Break down due to extreme heat, light, time, humidity, biological causes and presence of certain chemicals – STR analysis is better adapted to degraded DNA than RFLP ©2010 Elsevier, Inc. DNA DATABASE: THE FBI CODIS SYSTEM • Combined DNA Index System (CODIS) is the national database of DNA profiles administered by the FBI – Contains thousands of DNA profiles – Arranged in tiers: local, state, national – Contains three databases: forensic, criminal offenders, missing persons – Data consists of genotypes from 13 STR loci ©2010 Elsevier, Inc. DNA Case Backlog – Many states require some or all people arrested for crimes to be DNA typed – Caused huge backlog in forensic science laboratories ©2010 Elsevier, Inc. CHAPTER SUMMARY • DNA molecule is a double helix containing base pairs linked to a chemical backbone • DNA typing methods utilize differences in human genome to indentify people from biological evidence – RFLP – PCR • CODIS contains nearly six million profiles • Innocence Project uses DNA typing to re-examine criminal convictions • mtDNA is useful for typing old, degraded samples ©2010 Elsevier, Inc.