(Transcribed from Dr. Meissenburg’s handout by Brian Buschman)
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These notes are not like most in that they were not made from the lecture material but are only selected parts of the departmental handout. This handout will probably not be useful to anyone other then the author because I, for the sake of my study time, skipped many broad areas that I already know very well.
To carry out PCR you need the extracted DNA, dATP, dGTP, dCTP, dTTP and a heat stable DNA polymerase. It is possible to get a DNA polymerase that functions at 60 oC and is stable up to 90oC if you extract it from bacteria that can be found in the naturally boiling springs. The summary of the recipe is to:
· Mix all the stuff up.
· Denature the DNA by heating it to 90oC.
· Cool to 60oC for the DNA polymerase to function.
· Reheat to 90oC to start the process over.
· Do 20-30 cycles.
It is very useful to use this technique to amplify a small sample of DNA so that it can be analyzed by methods requiring more DNA then can be found in a couple of cells.
Hybrids of human and non-human cells are made which tend to spit out individual chromosomes. In cases where you can identify the gene you can use these organisms to determine the chromosome where the gene can be found. It is only good for determining which chromosome the gene is on, not where it is on the chromosome.
Bind a labeled probe to the desired sequence and look to see where the probe can be found.
Some diseases are caused by a deletion of a gene. If the deletion is large enough to detect you can use that to locate approximately where the gene can be found.
You locate where the gene is and then compare it to neighboring genes, if known. Usually genes that are close together will play similar roles in similar functions.
Identify genes based on how they are linked to other genes. There are multiple ways to judge that two genes are nearby.
1) Classical RFLP (restriction fragment length polymorphisms) are when a restriction site is altered by a mutation. You can tell which set of genes has the mutation because it will not be cleaved when it should be.
2) VNTR (variable numbers of tandem repeats) are when you can use the differing lengths of tandem repeats to give you clues as to what is going on.
There are various mathematical methods presented in the handout to figure out what is going on with it.
Cloning is simply inserting a chunk of DNA into a host for reproduction. It is used in the process of making a gene library.
A genomic library is when you insert the complete human genome into a number of cloning vectors (plasmids, l-phages or YAC’s) that are inserted into an antibiotic resistant bacteria. You then let them grow in the presence of the antibiotic. Then lyse them and blow the DNA onto nitrocellulose. This will give good set of genomic material on the gel.
cDNA is DNA made from mRNA by reverse transcriptase. You must be careful in what cells you use to isolate the mRNA because not all cells express genes in the same way. It is almost impossible to get a complete genomic library in this way because there are no cells that express the entire human genome and only genes that have been expressed will be reverse transcribed.
You place the cDNA in a bacteria with a strong promoter to express the gene. This only works with cDNA because bacteria are not capable of removing introns which always exist in human DNA.
The bacteria will secrete the given protein product. This is used to make synthetic versions of things like GH, factor VIII and insulin.
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