Tuesday, November 29, 2011

DNA sequencing: Southern blotting, Northern blotting, RT-PCR, microarray analysis and the difference between SNP and RFLP

DNA sequencing: Southern blotting, Northern blotting, RT-PCR, microarray analysis and the difference between SNP ("snip") and RFLP ("rif-flip").

DNA (deoxyribonucleic acid) Sequencing
Refers to several technologies that can be used to determin the the order of the nucleotide bases adenine, thymine, guanine, and cytosine in a DNA molecule. Usually this is used to discover the sequences for certain genes.

Southern Blotting
Is when three (or more) strands of DNA from different organisms are fragmented with the same restriction enzyme. The DNA is then loaded in to GEP chamber (gel electrophoresis) creating bands of different fragments of each set of DNA. The DNA is then transfered to a nitrocellulose membrane through the act of cappilary action when the membrane is placed on top of the agarose gel from the GEP. The DNA fragments in the nitrocellulose membrane then hybridize with radioactively labeled probe (single stranded DNA complimentary to only the gene of interest). A sheet of photographic paper (ie x ray paper) is then laid ontop of it showing only the fragments in the bands that binded with the probes giving and providing a visual image allowing the results to be interpreted. 

Northern Blotting
Uses the same processes as Southern blotting but instead of using DNA molecules, mRNA molecules from the organism are used. This techinque is commonly applied to track genes of interest at certain stages of embryonic developement.

RT-PCR (reverse transcriptase-polymerase chain reaction)
Similar to Northern blotting, RT-PCR begins with the restriction fragmentation through GEP of an organisms isolated mRNA. Resverse transcriptase is then added to create cDNA (complimentary DNA) which is then amplified through PCR using primers from the gene of interest. This techinque is commonly used to track genes of interest through embrionic stages of developement.

Microarray Analysis
The mRNA of a target sample is used to create cDNA with the addition of reverse transcriptase that is labeled radioactively. Then the cDNA mixture is applied to a microarray on which genes are fixed. Each spot in the slide indicates a different gene. Scan the microarray and each florescent spot represents the gene of interest expressed in the sample.

The difference between SNP and RFLP: SNP (single nucleotide polymorsphism) is a single base-pair site where varition is found in at least one percent of the population of interest. When a SNP is found that is presented in, say cancer patients, but not in unaffected patients; then the SNP can be studied and sequenced to [hopefully] better understand the disease (or sample/population) of interest. A RFLP (restriction fragment length polymorsphism) is a SNP that exists in the restriction site of an enzyme making the site unrecognizable and changing lengths of restriction fragments that are created when digested by that enzyme.