DNA Analysis Chapter 11 ©2010 Elsevier, Inc.

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 INTRODUCTION ©2010 Elsevier, Inc.

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 THE NATURE OF DNA ©2010 Elsevier, Inc.

– 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 Nuclear DNA ©2010 Elsevier, Inc.

– DNA molecule consists of sugar-phosphate backbones connected by linked base pairs: A-T, T- A, G-C, C-G Order of these pairs is controlled by genetic code, or blueprint that determines the characteristics of a person Nuclear DNA ©2010 Elsevier, Inc.

– 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 DNA in Cells ©2010 Elsevier, Inc.

DNA in Cells ©2010 Elsevier, Inc.

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 Genes and the Genetic Code ©2010 Elsevier, Inc.

– 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 Variations of Genes: Alleles ©2010 Elsevier, Inc.

– 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 Variations of Genes: Alleles ©2010 Elsevier, Inc.

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 Variations of Genes: Alleles ©2010 Elsevier, Inc.

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 Population Genetics ©2010 Elsevier, Inc.

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 Population Genetics ©2010 Elsevier, Inc.

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) DNA TYPING ©2010 Elsevier, Inc.

– 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. Restriction Fragment Length Polymorphism (RFLP) ©2010 Elsevier, Inc.

– 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 How RFLP Works ©2010 Elsevier, Inc.

– 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 How RFLP Works ©2010 Elsevier, Inc.

A technique known as Southern Blotting is used to transfer the separated DNA fragments onto a more rugged nylon membrane How RFLP Works ©2010 Elsevier, Inc.

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 Visualization of VNTRs ©2010 Elsevier, Inc.

Polymerase chain reaction is used for making copies of DNA using polymerase enzymes – Overcame issue of relatively large quantities required for RFLP The Polymerase Chain Reaction (PCR) ©2010 Elsevier, Inc.

– 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 The PCR Process ©2010 Elsevier, Inc.

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 – Cycle of three steps is repeated until sufficient DNA is produced; cycles The PCR Process ©2010 Elsevier, Inc.

The PCR Process ©2010 Elsevier, Inc.

– 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 DNA Typing of PCR Product ©2010 Elsevier, Inc.

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 Short Tandem Repeats (STRs) ©2010 Elsevier, Inc.

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 STRs ©2010 Elsevier, Inc.

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 STRs ©2010 Elsevier, Inc.

– 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 Gender Identification ©2010 Elsevier, Inc.

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 Mitochondrial DNA (mtDNA) ©2010 Elsevier, Inc.

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 Mitochondrial DNA (mtDNA) ©2010 Elsevier, Inc.

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 INTERPRETATION OF DNA TYPING RESULTS ©2010 Elsevier, Inc.

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 Comparison of DNA Samples ©2010 Elsevier, Inc.

– 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 Estimation of Population Frequencies ©2010 Elsevier, Inc.

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 Interpretation of DNA Typing Results: Purity Issues ©2010 Elsevier, Inc.

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 DNA DATABASE: THE FBI CODIS SYSTEM ©2010 Elsevier, Inc.

– Many states require some or all people arrested for crimes to be DNA typed – Caused huge backlog in forensic science laboratories DNA Case Backlog ©2010 Elsevier, Inc.

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 CHAPTER SUMMARY ©2010 Elsevier, Inc.