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Introduction to PCR lab In this experiment polymerase chain reaction (PCR) is used to amplify a short piece of DNA from the human chromosome 8. A short insertion of a DNA sequence, in this case Alu, which is found to be within the tissue plasminogen activator gene on chromosome 8, is observed very carefully. Even though the DNA from human to human does not differ a whole lot and has many similarities the diversity among regions of the human chromosomes is high. Because of the fact there is a variety a sequences of TPA- 25, it is said to be polymorphic meaning many forms are possible. This polymorphism helps us define the basis for diagnosis of genetic diseases, forensic identification, and paternity testing.
An Alu is a short interspersed, repetition of DNA elements that are scattered throughout the primate genome. The Alu in this element had a DNA sequence of 400 bp, which means if you insert the mixture of PCR and DNA and have the TPA 25 present into Agarose gel Electrophoresis a fragment line will form at 400 bp. It is called Alu after the single recognition site for the restriction enzyme Alu I, which is detected in the middle of the Alu element. The Alu element called TPA- 25 that is used in this experiment is located within an intron if the tissue plasminogen activator gene. Because the insertion of it is only present in some individuals and in other not we can get three main result. The individual can either be homozygous for the trait, heterozygous meaning one allele has it and the other one doesn t or homozygous but does not show the trait in either allele.
In this experiment PCR will do the job to detect whether a person is positive for the TPA- 25 insertion. PCR is a process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA. The DNA is then denatured to break the bonds between the strands of the DNA. When it cools, the primers bind to the separated strands, and DNA polymerase quickly builds a new strand by joining the free nucleotide bases to the primers. When this process is repeated, a strand that was formed with one primer binds to the other primer, resulting in a new strand that is restricted solely to the desired segment. Thus, the region of DNA between the primers is selectively replicated.
This lab process was developed in 1985 by Key B. Multi and used mostly in fingerprinting and in medical tests to identify diseases from the infectious agents DNA. The concept behind Electrophoresis is two identify the genotype of a particular gene, in this case TPA- 25. After a sequence has been amplified a sample of it is loaded into the wells of the Agarose gel along with a DNA weight marker and a an un amplified control of PCR. After Electrophoresis and staining the products will be visible as dark bands on the gel. By measuring how far each of these liquids traveled through the gel we can examine whether the person has the TPA- 25 insertion.
If two bands show up the person is said to be heterozygous meaning one allele has it the other doesn t. The control just gives us assurance that the PCR was not contaminated.
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