Forensic Science: The Basics DNA Typing Chapter 14 Jay A. Siegel,Ph.D. Power point presentation by Greg Galardi, Peru State College, Peru Nebraska Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. DNA Is better for identification than polymorphic substances due to: 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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) Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. DNA Only certain pairs of bases can join together No base can join itself A strand of DNA has millions of base pairs and rules can not be violated Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. Cellular DNA Within nucleus DNA is arranged in chromosomes Each human has 46 chromosomes Chromosomes are arranged in 23 pairs Each parent supplies one member of each of the 23 pairs Through chromosomes people inherit their physical, mental and emotional characteristics (continued) Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

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) Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. DNA Amplification Takes place in three steps; 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.

CRC Press, Jay A. Siegel Ph.D. 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 Presentation by Greg Galardi, Peru State College CRC Press, Jay A. Siegel Ph.D.