BCHM 307 1st Edition Lecture 27Outline of Last Lecture I. DNA AnalysisA. Gene CloningII. NucleasesA. EndonucleasesB. ExonucleasesC. Restriction EndonucleasesIII. DNA PlasmidsA. Plasmid PropertiesOutline of Current Lecture I. Polymerase Chain ReactionA. Taq PolymeraseII. DNA MutationsA. SNPsB. IndelsIII. Northern BlottingIV. MicroarraysCurrent LectureThis lecture looks at another way of performing DNA analysis. Polymerase chain reactions (PCR) can be used to synthesize, and amplify, specific segments of DNA. The advantage of using PCR is that you can start with a very crude sample of DNA. PCR works by using extracting the DNA and a small amount is added to a tube. DNA primers, dNTPs and Taq polymerase is added. Taq polymerase is a heat stable DNA polymerase. It is extracted from a thermophilic bacterium.The mixture is heated to 94 degrees Celsius first. This separates the DNA strands. Then the mixture is heated at 55 degrees Celsius. This allows the primers to bind to the separated DNA strands. Finally, the mixture is heated to 72 degrees Celsius. This allows the polymerase to lengthen. The three steps are repeated between 20 and 40 times. Once enough DNA is obtained, it is stained with ethidium bromide. This allows the DNA to be visible. The DNA can then be used for analysis. The final topic of this chapter is DNA mutations, or polymorphisms. The first type is called single nucleotide polymorphisms, SNPs. This is simply a change in a single base pair. A These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.branch off of this type is called a restriction fragment length polymorphism. These mutations exist within a restriction site. Insertions or deletions are also possible. This is when a sequence of bases is inserted or deleted from the DNA strand. mRNA can also be analyzed. This is done by the process called Northern blotting. In this process the RNA is first extracted, electrophoresed, and then blotted onto a nylon membrane. The membrane is exposed to a radioactive DNA probe that corresponds to a particular gene. The probe is then hydrogen bonded to the mRNA strand. A washing is done to wash away the probes that did not bind. After a staining is done, the parts where the probe bound can be seen by autoradiography. The amount of radioactivity present indicates the level of gene expression. mRNA can also be analyzed using a technique involving microarrays. This technique measures the expression of thousands of genes at once. The mRNA is first extracted. It is then reverse transcribed and biochemically tagged. It is hybridized to DNA oligonucleotides which areattached to a chip. cDNAs from a different sample are hybridized to different chips. The tagged cDNA then hybridize to the DNA on the chip via hydrogen bonds. The chip is treated with a fluorescent antibody. This recognizes the biochemical tag on cDNAs. The hybridization level can then be measured. The fluorescence of each position on the microarray with correspond to the amount of mRNA for each
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