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Fs Ch 14
- 1. Chapter 14 Biological Stain Analysis: DNA
Notas do Editor
- The word blood refers to a highly complex mixture of cells, enzymes, proteins, and inorganic substances. Plasma, which is the fluid portion of blood, is composed principally of water. Red blood cells (erythrocytes), white blood cells (leukocytes), and platelets are the solid materials suspended in plasma. Antigens, usually proteins, are located on the surface of red blood cells and are responsible for blood-type characteristics.
- 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. 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. For every antigen there is a specific antibody that will react with it to form clumps known as agglutination. Thus, if serum containing anti-B is added to red blood cells carrying B antigen, they will immediately react.
- 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. Microcrystalline tests, such as the Takayama and Teichmann tests, depend on the addition of specific chemicals to the blood so that characteristic crystals will be formed.
- 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.
- 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.
- 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 unequivocally identified by either the presence of spermatozoa or of p30, a protein unique to seminal plasma. Forensic scientists can successfully link seminal material to an individual by DNA typing.
- The rape victim must undergo a medical examination as soon as possible after the assault. At that time the appropriate items of physical evidence including clothing, hairs, and vaginal and rectal swabs can be collected for subsequent laboratory examination. 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.
- Suspect: -24 hours -ble 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.
- Portions of the DNA structure are as unique to each individual as fingerprints. The gene is the fundamental unit of heredity. Each gene is actually composed of DNA specifically designed to carry the task of controlling the genetic traits of our cells. The genes are positioned on chromosomes , threadlike bodies that appear in the nucleus of every body cell
- The egg cell from the mother always contains a long chromosome known as the X chromosome . The sperm cell from the father may contain either a long X chromosome or a short Y chromosome . When an X-carrying sperm fertilizes an egg, the new cell is XX and develops into a female. A Y-carrying sperm produces an XY fertilized egg and develops into a male.
- The position a gene occupies on a chromosome is its locus . Alternative forms of genes that influence a given characteristic and are aligned with one another on a chromosome pair are known as alleles . A gene pair made up of two similar alleles is said to be homozygous . A gene pair made up of two different alleles is said to be heterozygous .
- DNA is constructed as 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 bases on each strand are properly aligned in a double-helix configuration, which is two strands of DNA coiled together. As a result, adenine pairs with thymine and guanine pairs with cytosine. This concept is known as base pairing.
- 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. 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. The ability to multiply small bits of DNA by PCR now means that sample size is no longer a limitation in characterizing DNA recovered at a crime scene.
- 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. 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 are added to rebuild each of the separated strands. Now, this process is repeated 25 to 30 times.
- Portions of the DNA molecule contain sequences of bases that are repeated numerous times, known as tandem repeats. To a forensic scientist, these tandem repeats offer a means of distinguishing one individual from another through DNA typing. Tandem repeats seem to act as filler or spacers between the coding regions of DNA. What is important to understand is that all humans have the same type of repeats, but there is tremendous variation in the number of repeats each of us have.
- 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 sequences that repeat themselves within the DNA molecule. They serve as useful markers for identification because they are found in great abundance throughout the human genome.
- 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.
- 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.
- 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.
- Capillary electrophoresis separates DNA fragments on a thin glass column. Each end of the column is immersed in a reservoir of buffer liquid with electrodes. The DNA-containing sample solution is injected into one end of the column. The STR fragments move through the electrified column at a speed that is related to the length of the STR fragments. As the DNA peaks pass through the detector, they are recorded on a display known as an electropherogram.
- 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.
- 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. A single mitochondria contains several loops of DNA.
- 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 have been found to be highly variable and a procedure known as sequencing is used to determine the order of base pairs.
- 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.
- 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. Clothing from victim and suspect with blood evidence must be collected. 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.
- Each stained article should be packaged separately in a paper bag or in a well-ventilated box. 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. All biological evidence should be refrigerated or stored in a cool location until delivery to the laboratory. Standard/reference DNA specimens must also be collected, such as blood or the buccal swab (swabbing the mouth and cheek).