Class Name, Instructor Name Chapter 15: Biological Stain Analysis: DNA Class Name, Instructor Name Date, Semester 1

Nature of Blood Plasma, which is the fluid portion of blood, is composed principally of water and accounts for 55% of blood content. Red blood cells (erythrocytes), white blood cells (leukocytes), and platelets are the solid materials suspended in plasma. Antigens, usually proteins and carbohydrates, are located on the surface of red blood cells and are responsible for blood-type characteristics.

Nature of Blood Plasma contains proteins known as antibodies. The fundamental principle of blood typing is that for every antigen there exists a specific antibody. Antibodies are normally bivalent—that is they have two reactive sites. This means that an antibody can attach to antigens located on two red blood cells forming a network of cross-linked cells seen as clumping or agglutination.

Blood Typing More than 15 blood antigen systems have been identified, but the A-B-O and Rh systems are the most important. An individual that is type A has A antigens on his/her red blood cells, type B has B antigens, AB has both A and B antigens, and type O has neither A nor B antigens. Approximately 43% of the population is type O, 42% type A, 12% type B, and 3% type AB. Type A blood has only anti-B and no anti-A. Type B blood has only anti-A and no anti-B. Type AB blood has neither anti-A nor anti-B. Type O blood has both anti-A and anti-B. Rh factor is determined by the presence of another antigen, the D antigen. People having the D antigen are Rh positive; those not having the antigen are Rh negative.

Immunoassay The concept of specific antigen-antibody reactions has been applied to immunoassay techniques for detecting drugs-of-abuse in blood and urine. Antibodies that react with drugs do not exist naturally; however, they can be produced in animals by combining the drug with a protein and injecting this combination into the animal.

Immunoassay This drug–protein complex acts as an antigen stimulating the animal to produce antibodies which can be recovered in the blood serum of the animal. The recovered blood serum of the animal contains antibodies that are nearly specific to the drug. and is used to test blood or urine for the presence of a specific drug. Immunoassay is only presumptive in nature and its results must be confirmed by additional testing.

Forensics of Blood The criminalist must be prepared to answer the following questions when examining dried blood: Is it blood? From what species did the blood originate? If the blood is of human origin, how closely can it be associated to a particular individual? The determination of blood is best made by means of a preliminary color test.

Blood Color Tests A positive result from the Kastle-Meyer color test is highly indicative of blood. Hemoglobin causes a deep pink color. Alternatively, the luminol test is used to search out trace amounts of blood located at crime scenes. Produces light (luminescence) in a darkened area. Luminol is extremely sensitive and is capable of detedting blood that has been diluted up to 100,000 times.

Blood Origin Testing Once the stain has been characterized as blood, the precipitin test will determine whether the stain is of human or animal origin. The precipitin test uses antisera normally derived from rabbits that have been injected with the blood of a known animal to determine the species origin of a questioned bloodstain. The technique of gel diffusion takes advantage of the fact that antibodies and antigens diffuse or move toward one another on an agar plate. The extracted bloodstain and the human antiserum are placed in separate holes opposite each other on the gel. If the blood is human, a line of precipitation forms where the antigens and antibodies meet.

Blood Origin Testing Once the stain has been characterized as blood, the precipitin test will determine whether the stain is of human or animal origin. The precipitin test uses antisera normally derived from rabbits that have been injected with the blood of a known animal to determine the species origin of a questioned bloodstain. The technique of gel diffusion takes advantage of the fact that antibodies and antigens diffuse or move toward one another on an agar plate. The extracted bloodstain and the human antiserum are placed in separate holes opposite each other on the gel. If the blood is human, a line of precipitation forms where the antigens and antibodies meet.

Individualizing Bloodstains Once it has been determined that the bloodstain is of human origin, an effort must be made to associate or dissociate the stain with a particular individual. DNA analysis has allowed forensic scientists to associate blood to a single individual.

Testing for Seminal Stains Many of the cases sent to a forensic laboratory involve sexual offenses, making it necessary to examine exhibits for the presence of seminal stains. The best way to locate and at the same time characterize a seminal stain is to perform the acid phosphatase (an enzyme secreted into seminal fluid) color test. A purple color indicates acid phosphatase enzyme. Semen can be identified by either the presence of spermatozoa or p30, a protein present in seminal plasma. Forensic scientists can successfully link seminal material to an individual by DNA typing.

Sexual Assault Evidence The sexual assault victim must undergo a medical examination as soon as possible after the assault. At that time the appropriate items of physical evidence are collected: Pubic Combings Oral Swabs Pubic Hair Controls (15-20) Head hair Controls (50) External Genital Swabs Blood Vaginal Swabs Buccal Swab Cervix Swabs Fingernail Scrapings Rectal Swab Urine Specimen

Sexual Assault Evidence All outer and undergarments should be carefully removed and packaged separately in paper (not plastic) bags. Bedding, or the object upon which the assault took place, may also be carefully collected.

Sexual Assault Evidence If a suspect is apprehended within 24 hours of the assault, it may be possible to detect the victim’s DNA on the male’s underwear or on a penile swab of the suspect. Items routinely collected from the suspect include all clothing, pubic hair, head hair, penile swab, and a blood sample or buccal swab for DNA typing. The forceful physical contact between victim and assailant may result in a transfer of such physical evidence of blood, semen, saliva, hairs, and fibers.

Genes and Chromosomes The gene is the basic unit of heredity. A chromosome is a threadlike structure in the cell nucleus along which the genes are located. Each gene is actually composed of DNA specifically designed to carry the task of controlling the genetic traits of our cells. The position a gene occupies on a chromosome in known as a locus. Approximately 30,000 human genes have been identified. Most human cells contain 46 chromosomes, arranged in 23 mated pairs. The only exceptions are the human reproductive cells, the egg and sperm, which contain 23 unmated chromosomes. During fertilization, a sperm and an egg combine so that each contributes 23 chromosomes to form the new cell, or zygote, that develops into the offspring.

Genes and Chromosomes An allele is any of several alternative forms of genes at a particular locus and that are aligned with one another on a chromosome pair. A heterozygous gene pair is made up of two different alleles; a homozygous gene pair is made up of two similar alleles. When two different genes are inherited, the characteristic coded for by a dominant gene will be expressed. The characteristic coded for by a recessive gene will remain hidden.

The Structure of Nuclear DNA Actually, a human cell contains two types of DNA-nuclear and mitochondrial. We will focus first on nuclear DNA. Nuclear DNA strand is a very large molecule made by linking a series of repeating units called nucleotides. A nucleotide is composed of a sugar, a phosphorous-containing group, and a nitrogen-containing molecule called a base. Four types of bases are associated with the DNA structure: adenine (A), guanine (G), cytosine (C), and thymine (T).

The Structure of Nuclear DNA The DNA molecule is comprised of two strands each wrapped around the other in the form of a double-helix The bases on each strand are properly aligned in a manner known as complementary-base pairing. As a result, adenine pairs with thymine and guanine pairs with cytosine.

DNA Replication DNA duplicates itself prior to cell division. DNA replication begins with the unwinding of the DNA strands of the double helix. Each strand is now exposed to a collection of free nucleotides that will be used to recreate the double helix, letter by letter, using base pairing. Many enzymes and proteins, such as DNA polymerases, are involved in unwinding the DNA, keeping the DNA strands apart, and assembling the new DNA strands in the proper order (A with T and G with C).

Polymerase Chain Reaction (PCR) Testing Polymerase chain reaction (PCR) is a technique for replicating small quantities of DNA or broken pieces of DNA found at a crime scene, outside a living cell. Polymerase chain reaction is the outgrowth of knowledge gained from an understanding of how DNA strands naturally replicate within a cell. For the forensic scientist, PCR offers a distinct advantage in that it can amplify minute quantities of DNA many millions of times.

Polymerase Chain Reaction (PCR) Testing First, the DNA is heated to separate it. Second, primers (short strands of DNA used to target specific regions of DNA for replication) are added, which hybridize with the strands. Third, DNA polymerase and free nucleotides (A, T, G, and C) are added to rebuild each of the separated strands. Now, this cycle is repeated 25 to 30 times. Within a few hours a short strand of DNA can be multiplied a billionfold.

DNA Tandem Repeats Portions of the DNA molecule contain sequences of bases that are repeated numerous times, known as tandem repeats. What distinguishes one tandem repeat from another is how the ATGCs repeat themselves in the tandem’s core. Tandem repeats seem to act as filler or spacers between the coding regions of DNA. To a forensic scientist, these tandem repeats offer a means of distinguishing one individual from another through DNA typing. What is important to understand is that for a particular tandem there is tremendous variation in the number of repeats each of us have for that tandem.

Short Tandem Repeats (STRs) The latest method of DNA typing, short tandem repeat (STR) analysis, has emerged as the most successful and widely used DNA profiling procedure. STRs are locations on the chromosome that contain short tandem repeats that repeat themselves within the DNA molecule.

STR Advantages STRs normally consist of repeating sequences of 3 to 7 bases in length, and the entire strand of an STR is also very short, less than 450 bases in length. This means that STRs are much less susceptible to degradation and may often be recovered from bodies or stains that have been subjected to extreme decomposition. Also, because of their shortness, STRs are ideal candidates for multiplication by PCR, thus overcoming the previously mentioned limited-sample-size problem often associated with crime-scene evidence. STRs usually produce a two-band pattern;, thus interpretation of mixtures is simplified. An STR pattern arising from two individuals will have four peaks.

Typing STRs Let’s look at commonly used STR known as TH01. It contains the repeating sequence A-A-T-G. Seven TH01 variants have been identified in the human genome, these variants contain 5 to 11 repeats of A-AT-G. Two such variants contain 6 repeats of A-A-T-G and 8 repeats of A-A-T-G. During a forensic examination, TH01 is extracted, amplified by PCR, and separated by electrophoresis.

Typing STRs By examining the distance the STR has migrated on the electrophoretic plate, one can determine the number of A-T-T-G repeats in the STR. Typically, every person has two STR types for TH01, one inherited from each parent. The DNA type known as TH01 6,8 is found in approximately 3.5% of the population

The Power of STR What makes STRs so attractive to forensic scientists is that hundreds of different types of STRs are found in human genes. The more STRs one can characterize, the smaller will be the percentage of the population from which a particular combination of STRs can emanate. This gives rise to the concept of multiplexing. Using the technology of PCR, one can simultaneously extract and amplify a combination of different STRs.

Standardizing STR Testing Currently, U.S. crime laboratories have standardized on 13 STRs for entry into a national database (CODIS). A high degree of discrimination and even individualization can be attained by analyzing a combination of STRs (multiplexing) and determining the product of their frequencies. With STR, as little as 125 picograms of DNA is required for analysis. This is equivalent to about 18 DNA containing cells.

Sex Determination by STRs The amelogenin gene, which is actually the gene for tooth pulp, is shorter by six bases in the X chromosome than in the Y chromosome. Hence, when the amelogenin gene is amplified by PCR and separated by electrophoresis, males, who have an X and a Y chromosome, show two bands; females, who have two X chromosomes, have just one band. Forensic scientists can also type STRs located on the Y or male chromosome. Y-STRs prove useful when multiple males are involved in a sexual assault. For example an STR pattern arising from three males will have only three bands.

Mitochondrial DNA Another type of DNA used for individual characterization is mitochondrial DNA. Mitochondrial DNA (mtDNA) is located outside the cell’s nucleus and is inherited from the mother. Mitochondria are structures found in all our cells used to provide energy that our bodies need to function.

Mitochondrial DNA Testing Mitochondrial DNA typing does not approach STR analysis in its discrimination power and thus is best reserved for samples, such as hair, for which STR analysis may not be possible. Forensic analysis of mtDNA is more rigorous, time consuming, and costly when compared to nuclear DNA analysis. Also, all individuals of the same maternal lineage will be indistinguishable by mtDNA analysis. Two regions of mtDNA HV 1 and HV 2, have been found to be highly variable and a procedure known as sequencing is used to determine the order of base pairs.

CODIS Perhaps the most significant tool to arise from DNA typing is the ability to compare DNA types recovered from crime scene evidence to those of convicted sex offenders and other convicted criminals. CODIS (Combined DNA Index System) is a computer software program developed by the FBI that maintains local, state, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, and profiles of missing persons. It currently contains about 350,000 profiles from unsolved cases. Currently, U.S. crime laboratories have standardized on 13 STRs for entry into a national database (CODIS).

Collection of DNA Evidence Sources of DNA: Blood Skin Cells (touch DNA) Semen Hair Saliva Bone DNA is a powerful creator of physical evidence at crime scenes; e.g., bottles, cans, glasses, cigarettes, bite marks, and envelopes.

Collection of DNA Evidence Clothing from victim and suspect with blood evidence must be collected. Dried blood is best removed from a surface by using a sterile cotton swab lightly moistened with distilled water that is air dried before being placed in a swab box, then a paper or manila envelope. Standard/reference DNA specimens must also be collected, such as blood or the buccal swab (swabbing the mouth and cheek).

Packaging Biological Evidence Before the collection of biological evidence begins, it is important that it be photographed and recorded on sketches. Wearing disposable latex gloves while handling the evidence is required. The packaging of biological evidence in plastic or airtight containers must be avoided because the accumulation of residual moisture could contribute to the growth of DNA-destroying bacteria and fungi.

Packaging Biological Evidence Each stained article should be packaged separately in a paper bag or in a well-ventilated box. All biological evidence should be refrigerated or stored in a cool location until delivery to the laboratory.