Lab Report N*8 Genetic-Fingerprinting PDF

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Laboratory reports for Spring 2018 Cell & Molecular Biology Laboratory by Professor. Henri P. Antikainen and Cervantes. ...

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Genetic Fingerprinting Alu Element – TPA 25 Introduction: The DNA sequences of the human genome are nearly identical among individuals. Although the DNA from different individuals is more alike than different, in many regions of the DNA, chromosomes exhibit a great deal of diversity, known as polymorphic. Polymorphic sequences provide diagnosis for inherited diseases, forensic identification, and paternity testing. PCR is based on using the ability of DNA polymerase to synthesize a new strand of DNA complementary to the given template strand. Since DNA polymerase can add nucleotides only onto a preexisting 3’ -OH group, it needs a primer to which it can add the first nucleotide. PCR primers are short fragments of the single stranded DNA that are complementary to DNA sequences to flank that specific targeted region, as the polymerase adds dNTPs. Primers make it possible to delineate a specific region of the template for amplification. As PCR or Polymerase-chain reaction is used to amplify small portions of the DNA from different chromosomes through a cycle of three functions at different temperatures: denaturing (94-95 *C), annealing (50-56 *C), and extending stage (72 *C), at the end of the PCR reaction, the specific sequence will be accumulated in billions of copies, known as amplicons. The number of copies of DNA synthesized is given by y = 2x where x is given by the number of reaction cycles. Overall, this type of cycle is an exponential amplification Other important components of the PCR reaction are: RT-PCR (building blocks of the new DNA strand), Reverse transcriptase (cDNA with enzyme), and DNA template (the sample DNA that contains the targeted sequence). In this experiment, we will amplify the insertion of the Alu DNA sequence in the TPA gene. This Alu element, called TPA-25, is of dimorphic nature, meaning that it is only present in some individuals. The Alu element is insertion occurs within an intron (a region of the gene whose transcribed RNA is eliminated before translation), since it will not affect the production of a complete TPA region. The element is present in amplified regions of 400-bp fragment and not present in 100-bp long fragments. To clearly measure this there are three possible genotypes: Homozygotes – presence only on the 400-bp fragment (+ /+) Homozygotes – absence only on the 400-bp fragment (- /-) Heterozygotes – producing both 400-bp and 100-bp fragments (+ /-) Objective: We will use PCR to look for an insertion of a short nucleotide sequence called Alu, which is a sequence that tends to appear within the tissue of a plasminogen activator (TPA) gene.

Methods: There are 2 steps to this experiment: A) Collection an amplification of DNA - Isolation of Cheek Cell DNA for PCR Amplification B) Determining the TPA-25 genotype - Population genetics - Practical applications in Forensic science Obtained a template DNA, from our copies of chromosome 8 by saline mouthwash, a painless, bloodless and noninvasive procedure. The cells were collected by centrifugation and suspended in a solution containing the matrix Chelex, which binds the metal ions that inhibit the PCR reaction. The cells were then lyzed by boiling and centrifuged to remove the cells debris. A sample of the supernatant containing chromosomal DNA was mixed with Taq DNA polymerase, oligonucleotide primers, and the dNTPS building blocks with a co-factor of Mg2+ as (MgCl2). The temperature cycling was used to denature the targeted DNA, anneal the primers and extend a complementary DNA strand through the PCR cycle. Finally a electrophoresis and staining of the amplification was done to create the distinct bands in the gels. The distance of the gel allows us to distinguish whether or not the production of the TPA-25 insertion is inversely proportional to the presence or absence by movement. Results: This photograph represents the end of the electrophoretic run, which created our sample stained gel to determine whether or not the individual is a carrier of the Alu element TPA-25.

Discussion: We investigated the sequence of Alu element, TPA-25 through the process of PCR reaction. The DNA sequences of the human genome are nearly identical among different individuals. Although in different regions of the DNA, chromosomes exhibit a great deal of diversity, known as polymorphic which provide diagnosis for inherited diseases, forensic identification, and paternity testing. PCR or Polymerase-chain reaction is used to amplify small portions of the DNA from different chromosomes through a cycle of three functions at different temperatures: denaturing (94-95 *C), annealing (50-56 *C), and extending stage (72 *C). By amplifying the insertion of the Alu DNA sequence in the TPA gene, we determine whether one was a homozygote or heterozygote of the allele. This Alu element, called TPA-25, is of dimorphic nature, meaning that it is only present in some individuals. The Alu element is insertion occurs within an intron (a region of the gene whose transcribed RNA is eliminated before translation), since it will not affect the production of a complete TPA region. The element is present in amplified regions of 400bp fragment and not present in 100-bp long fragments. Interpreting the DNA a. TPA-25 Genotype determination: The genotype of the student sample provided to us is heterozygous for the TPA-25 Alu insertion (+/-) since there is two visible bright bands that are likely to be the true alleles. b. Population Genetics: Unable to answer questions as the class used the same allele genotype. c. Practical and Applications: The practical problem of using finger-printing in criminal investigation is that it’s not convincing at proving guilt since many people could have the same exact combination. This could result in the imprisonment of innocent people. However, if the suspect does not have the allele combinations present at the crime scene, it would be difficult for investigator to determine whether or not the person is the suspect, then there would have to be first-eye witnesses to the events. Conclusion: In conclusion we used PCR to determine if an insertion of a short nucleotide sequence called Alu element, TPA-25 was found within the DNA sample taken by saline mouthwash procedure. The TPA-25 gene/protein is found within the tissue of plasminogen activator (TPA) gene. Overall, we understand the different principles and usages of DNA fingerprinting and how it’s used in different fields, such as in biological sciences, laboratory and forensic science....