Structural Biochemistry/DNA recombinant techniques/Northern Blot

From Wikibooks, open books for an open world
Jump to: navigation, search

Introduction[edit]

The Northern blot, also known as the RNA blot, is one of the blotting techniques used to transfer DNA and RNA onto a carrier for sorting and identification. The Northern blot is similar to the Southern blot except that RNA instead of DNA is the subject of analysis in this technique. First, the RNA from each tissue must be purified so that it can be examined for expression of the gene under investigation. The RNA samples are then loaded onto an agarose gel. The gel is then subjected to an electric current that causes the RNA in each sample to migrate toward the bottom of the gel. Smaller RNAs move faster while larger RNAs migrate more slowly. This separation process is known as electrophoresis. The separated RNA fragments are then blotted onto a special filter paper so that each RNA molecule retains its position relative to all the other molecules. The filter is then exposed to radioactive probes that hybridize to complementary sequences in the blot. The filter is then placed on a film for autoradiography and the film is developed. Lastly, a band should be observed on the autoradiograph if the probe has hybridized to a stretch of RNA on the filter. A band will only be visible in the column containing a tissue where the gene represented by the probe has been expressed.

The Northern blot is useful for the study of gene expression in two ways. First, the position of bands on the blot provides a direct measure of RNA size. Knowing the size of the RNA will provide an estimate for the transcript’s coding capacity and thus the size of the protein it encodes. Second, the Northern blot analysis of RNA samples from many different tissues enables researchers to determine which specific tissue a gene is expressed in along with the relative levels of its expression in all cells where transcription is occurring. The Northern blot is a valuable method used by researchers in determining gene expression patterns. For example, many scientists researching Huntington disease or breast cancer are able to determine the expression patterns of the genes responsible for these diseases using blotting techniques. The three blotting techniques including the Northern blot have proven to be important and positive advancements in science.

Northern Blot is a derivative of the Southern blot, which, like the Southern Blot, utilizes electrophoresis separation analysis and a hybridization probe for detection. For both techniques, the hybridization probe for detection can be made from either DNA or RNA. The main difference between the two techniques is that the northern Blot is performed to study gene expression by analyzing RNA instead of DNA. There are 3 types of RNA: tRNA (transfer RNA - active in assembly of polypeptide chains), rRNA (ribosomal RNA - part of the structure of ribosomes) and mRNA (messenger RNA - the product of DNA transcription and used for translation of a gene into a protein). It is mRNA which is isolated and hybridized in northern blots.The formaldehyde was use in electrophoresis gel as a denaturant because the sodium hydroxide treatment used in the Southern blot procedure would degrade the RNA. The northern Blot was developed at Stanford University in 1977 by James Alwine, David Kemp, and George Stark.

Process[edit]

The steps of Northern Blotting are as follows: Northern.jpg

1) RNA isolation: mRNA is extracted from the cells and purified.

2) Probe generation: The mRNA is loaded onto a gel for electrophoresis. Lane 1 has size standards (a mix of known RNA fragments) Lane 2 has the RNA.

3) Denaturing agarose gel electrophoresis: An electric current is passed through the gel and the RNA moves away from the negative electrode. The distance moved depends on the size of the RNA fragment. Since genes are different sizes the size of the mRNAs varies also. This results in a smear on a gel. Standards are used to quantitate the size. The RNA can be visualized by staining first with a fluorescent dye and then lighting with UV.

4) Transfer to solid support and immobilization: RNA is single-stranded, so it can be transferred out of the gel and onto a membrane without any further treatment. The transfer can be done electrically or by capillary action with a high salt solution.

5) Prehybridization and hybridization with probe: A labelled probe specific for the RNA fragment in question is incubated with the blot. The blot is washed to remove non-specifically bount probe and then a development step allows visualization of the probe that is bound.

6) Washing: The probe is bound specifically to the target mRNA and that there is negligible non-specific binding to other mRNA or the nylon membrane itself.

7) Detection: Hybridization signals are then detected.

8) Stripping and reprobing (optional):

RNA Isolation[edit]

This part of the Northern Blot is an important step because high quality, intact RNA needs to be obtained. There are several ways the isolation can be performed; however, common attributes include cellular lysis and membrane disruption, inhibition of ribonuclease activity, deporoteinization, and recovery of the intact RNA.

Probe Generation[edit]

Although the Southern blot is named after Edwin Southern [1], it is counter-intuitive to learn that the Northern blot was developed by James Alwine, David Kemp, and George Stark [2]. The name 'Northern Blot' is merely a play on words. Southern, Northern, and Western blots are known respectively for their analysis of DNA, RNA, and proteins, respectively.

Western Blot and Others[edit]

Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples.


Procedure:

a) Proteins are separated by gel electrophoresis, usually with SDS-PAGE to have all the proteins carries negative charge(s).

b) The proteins then transferred to a sheet of special blotting paper called nitrocellulose sheet although other types of paper or membranes can be used. The proteins retain in the same pattern of separation they had previous on the gel.

c) The blot is incubated with a generic protein (e.g. milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it which cannot be seen at this time.

d) The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product, which can be seen and photographed.

Add caption here


Other similar techniques such as far-western blots and south-western blots allow scientists to examine protein-protein interactions and DNA-protein interactions respectively.

References[edit]

  1. Berg, Jeremy M. (2007). Biochemistry (6th Ed. ed.). W. H. Freeman and Company. ISBN 0-7167-8724-5. 
  2. Alwine JC, Kemp DJ, Stark GR (1977). "Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes". Proc. Natl. Acad. Sci. U.S.A. 74 (12): 5350–4. doi:10.1073/pnas.74.12.5350. PMID 414220. 

3. Ambion. Applied Biosystems. http://www.ambion.com/techlib/basics/northerns/index.html