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Sequence Analysis of Single Polymorphism in the Factor V Gene - page 2 / 15





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Biol  420                                                                                                                                          Fall 2007

Polymerase chain reaction and DNA sequencing are two techniques commonly used to detect these types of single base polymorphisms.  

What is the Polymerase Chain Reaction?  PCR is a technique used to amplify very small amounts of DNA for various uses such as DNA fingerprinting and genetic testing.  It allows for selective amplification of a specific sequence of DNA.  Millions of copies of a region of DNA or a gene can be produced using PCR.  PCR allows amplification of your gene of interest to occur through the use of  a heat stable DNA polymerase called Taq polymerase and two primers (DNA sequences that are specific to a particular region of DNA) that hybridize to opposite strands of DNA, flanking the region of interest in the target or template DNA.   In a series of heating and cooling cycles, the DNA polymerase uses the primers as a template to generate numerous copies of the region of DNA between the primer sequences.

What is DNA Sequencing?  (Also see p. 125-26 of your textbook, The Cell:  A Molecular Approach) Sanger sequencing is a method of determining the base pair sequence of a gene or portion of a gene.  It is called Sanger sequencing after the individual the devised the method of replication using chain terminating dideoxy nucleotides in 1974.  By doing this you generate a series of fragments each one base longer than the last.  Then you can analyze the size of your DNA fragments using gel electrophoresis.

With the many advancements in technology since 1974, the Sanger method has become outdated. However, the new technology that has emerged to replace this method is based on the same principles of Sanger's method. Automated sequencing has been developed so that more DNA can be sequenced in a shorter period of time. With the automated procedures the reactions are performed in a single tube containing all four ddNTP's, each labeled with a different color dye (Russell, 2002).

Figure 1: In automated sequencing, the oligonucleotide primers can be "end-labeled" with different color dyes, one for each ddNTP. These dyes fluoresce at different wavelengths, which are read via a machine (Metzenberg).

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