Module 5: Molecular Cell Biology

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Northern Blot

Back 1

-mRNA is isolated from the tissue of interest and separated by size on an agarose gel. It is then blotted onto the membrane and probed.
-Applications: to determine which tissues express a gene, under what conditions the gene is expressed, and how much RNA is made. (can tell what tissue, but not which specific cell types).

Front 2

Western Blot

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-the proteins are separated on an acrylamide gel and transferred to membrane. In a Western blot, the membrane is then probed with an antibody that can bind the protein of interest.
-Applications: determines where and when proteins are present.

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Southern Blot

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-enables one to test for the presence of homologous genes without having to create a library. In a Southern blot, the DNA to be probed is cut with restriction enzymes, and the DNA pieces are separated by size by electrophoresis. Once the DNA is separated, it is transferred, or blotted, to a membrane. This membrane can then be probed the same way as the membranes used to screen libraries.
-Applications: whether a gene has multiple copies in the nucleus, presence of related genes, how conserved 2 genes are, and as a diagnostic tool in genetic diseases.

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in situ hybridizations

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-tissues are fixed or frozen under conditions in which the RNA remains in position. Often, the tissue is then sectioned into small slices and mounted on slides. The tissue is then treated to produce small holes that will allow a probe to enter the cells. The probe is added and hybridizes with the RNA in the tissue; then excess probe is washed out. To visualize the presence of the probe, a thin photographic film can be layered on the tissue and, after exposure in the dark, developed. Both the tissue and the silver grains of the film can then be seen under the microscope and the location of the RNA within the tissue can be determined.
-Application: localization of DNA (or proteins) to chromosomes in karyotype analysis.

Front 5

the techniques and use of sequencing

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-Use: to determine DNA base sequence. With the sequence, we can predict the protein sequence, look for regulatory regions, analyze the processing of the message, etc.
-Techniques (2 primary):
Maxam and Gilbert chemical method
Sanger and Barrell enzymatic method (more popular): This method uses interrupted DNA synthesis to determine the sequence. The DNA to be sequenced is divided into four tubes. Each tube contains the DNA, DNA polymerase, normal nucleotides, a labeled primer complementary to one end of the DNA, and a small amount of one of four abnormal nucleotides, called dideoxyribonucleoside triphosphates. The DNA polymerase begins to extend the primer, usually incorporating normal nucleotides into the elongating DNA strand.

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the techniques and use of footprinting

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-Use: tells us about the regulatory regions of DNA. (sequences recognized by regulatory proteins)
-Techhnique: If pure, labeled DNA molecules are treated with nuclease so that, on average, only one cleavage is made in each DNA molecule, a series of DNA fragments will be produced. When separated on an acrylamide gel, they will produce a ladder, with each band one nucleotide longer than the next. If the same procedure is performed with DNA bound to a DNA binding protein, the nuclease will be prevented from cutting the DNA in those regions that are protected by the protein. The DNA fragments of those sizes will be missing; on the gel they appear as a band-free region called a footprint