Link to our lab as a pdf - College of Science - Marshall University

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Forensic DNA Technology sequence at that site, preventing a cut. The lengths of DNA between the cut sites vary because of these mutations. The varying lengths of DNA fragments reflect the uniqueness of the DNA sample, and are compared to fragment size patterns generated from other DNA samples. RFLP analysis requires a large amount of intact DNA to obtain a meaningful result. While some crime scenes have much DNA-containing evidence, most contain only a small amount of material to work with, limiting the usefulness of RFLP analysis. The ability of forensic analysts to copy small amounts of DNA into larger quantities is critical when faced with crime scenes with little biological evidence – a cash register that has only been touched by a suspect, for example. It is also likely that some or all of the biological material found at a crime scene has been exposed to environment degradation in the form of weather, time, or the sun’s UV rays. The polymerase chain reaction (PCR) is a laboratory technique that mimics the biological process by which DNA replicates within a cell. Unlike RFLP which cuts DNA apart to produce an identifying pattern, PCR makes many copies of a selected region of junk DNA that has variable length within the population. The identifying pattern is created when the collective lengths of eight to sixteen of these regions, called short tandem repeats (STR), are compared. Figure 1. Possible genotypes of offspring given the genotypes of the mother and father. STRs used in DNA typing are located on different chromosomes, and therefore follow Mendel’s laws of segregation and independent assortment. These laws assert that we receive exactly one STR allele from each parent, and that receiving any given allele in no way influences the alleles received for other STRs. STRs can be used for paternity testing, because half of the offspring’s alleles must come from one parent and the other half from the other parent. Figure 1 shows a mother with the 2 and 3 repeat alleles of an STR locus, while the father has the 3 and 6 repeat alleles. The Punnett square predicts the possible allele combinations for their children. The father can contribute either the 3- or the 6-repeat allele to his offspring, while the mother can contribute either the 2- or the 3-repeat allele. They could have four children with completely different STR genotypes. By chance alone, they could also have four children with the same genotype. However, they cannot have a child with a new allele, unless there was a mutation, which is very rare. Electrophoresis is used to separate DNA fragments by size. Electrophoresis takes advantage of fact that there are electrical charges carried by the phosphate backbone of the DNA molecule. Since DNA fragments are negatively charged, they migrate toward the positive pole when placed in an electric field. When DNA is forced to move through a separation gel matrix, fragments are sorted based on size because the gel prevents larger fragments from travelling as quickly as smaller fragments. The most common separation matrices used are agarose and acrylamide gels. Agarose gels are made from a component extracted from seaweed which is dissolved in a heated solution. When the solution cools, microscopic pores are formed that act as a molecular sieve through which DNA molecules pass. Think of the separation matrix as a kelp forest dense with fronds but having pockets of space in between. These spaces are much like the pores in the gel. Due to their size, small fish have an easier time navigat- The Mystery of Lyle and Louise 15
Forensic DNA Technology stabilizes the gel’s pH. This same solution is also used to submerge the gel, providing an electrically and chemically uniform medium for the process. Since the phosphate backbone of DNA only has a negative charge in neutral to basic solutions, the solution is pH buffered to prevent it from becoming acidic, which would cause the DNA to reverse direction and move toward the negative pole. Figure 2: gel visualization of genotypes. ing through the plants than do the larger fish. Both eventually get through, but the small one travels faster. Applying this analogy to a gel matrix, the DNA travels through a gel, and just as in the seaweed forest, smaller fragments travel faster than the larger. When, after a certain period of time, the electrical force that moves the DNA through the matrix is removed, the newly spread-out DNA fragments stop moving. Their position in the gel is a direct relation to their size and they can be stained and their sizes compared to one another. Figure 2 is a schematic of a stained gel of each of the four possible genotypes of children from the mother and father in Figure 1. Notice how there is only one band for the homozygous genotype, C, because both the mother and father contributed the same allele. When visualized, this band should be darker than the other bands because there is twice as much DNA in that area as in the other bands. In electrophoresis, the solution used to create an agarose gel contains a salt dissolved in water, normally either Tris-acetate-EDTA (TAE) or Tris-borate- EDTA (TBE), that promotes electrical current and 16 Following separation by electrophoresis, fragments can be visualized by using several methods that directly bind to the DNA molecule. The most common in an educational setting is to stain with methylene blue. After electrophoresis, the gel is soaked in a stain solution. The stain adheres to the DNA molecules, but is also absorbed into the pores of the gel. Because of this, the entire gel is blue until it is allowed to soak in clear water, which dilutes the stain absorbed into the gel; a process called destaining. The dye bound to the DNA molecules is not removed during de-staining, and may be seen with the naked eye. In commercial and research settings, ethidium bromide is the most common nucleic acid stain. However, it can only be visualized under UV light and is mutagenic, which makes it unsuitable for novices. In this lab, a proprietary formula called BlueVis is used. BlueVis has no special safety concerns and stains the DNA while it migrates on the gel, reducing the need for de-staining. Other visualization methods include radioactive labels and commercially available fluorescent dyes which can be incorporated during the PCR process. To confirm the size of each PCR product, DNA size ladders are run on the same gel as the unknown samples. These ladders contain a series of known purified DNA fragments corresponding to most of the known variant forms of each portion of target DNA; each variant form is called an allele. Therefore, these allelic ladders provide a means for determining the length, and thus the specific variant or allele type, of each amplified product for a given sample. Theoretically, a large number of alleles are possible for any given loci; however, generally only four to ten alleles are common within a population. The alleles lengths that are present on the ladder, The Mystery of Lyle and Louise
Page 1 and 2: An Identity Crisis A Lab on DNA Typ
Page 3 and 4: Table of Contents Introduction to I
Page 5 and 6: Introduction to Identity Crisis WEL
Page 7 and 8: These notes are provided to assist
Page 9 and 10: Glossary Agarose: A molecule purifi
Page 11: PCR Technology PCR is the cornersto
Page 15 and 16: 18 The Mystery of Lyle and Louise
Page 17 and 18: The Investigation this time, invest
Page 19 and 20: The Evidence Evidence was collected
Page 21 and 22: Pre-Lab Questions DNA Background 1.
Page 23 and 24: Lab Procedure 6. 7. 8. 9. to go dow
Page 25 and 26: Post-Lab Questions Short Answer 1.
Page 27: Mock Trial documents prepared by th

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