CA10 (2010) - Question E2

Describe the following molecular techniques and provide an example of their application in Oncology. Use diagrams to illustrate your answer.

  1. Microarray gene profiling
  2. RT-PCR

Curriculum Link: Molecular Techniques In Pathology

Reverse Transcriptase - Polymerase Chain Reaction

Polymerase chain reaction is the use of progressive heating and cooling of DNA, together with DNA polymerase, to amplify the amount of DNA in a nucleus. This technique only works with DNA, and not RNA, and therefore reverse transcriptase - PCR was designed to convert RNA to a DNA molecule, allowing normal PCR to be performed.

Steps of RT PCR

Reverse Transcriptase

The RNA molecules under investigation are mixed with the enzyme reverse transcriptase. This enzyme assembles a complementary strand of DNA from the RNA molecule (cDNA).

Polymerase Chain Reaction

The newly created cDNA then undergoes polymerase chain reaction. This requires the repetition of several steps:

  • Application of DNA primers and DNA polymerase to the solution
  • Heating of the DNA strands to dissociate them from each other
  • Cooling of the DNA strands, allowing the DNA primers to assemble next to the target sequence of interest (forming a short dsDNA segment with a long ssDNA strand attached
  • Waiting for DNA polymerase to assemble a complementary strand of DNA, extending from the primer along the DNA sequence of interest
  • Heating of the DNA strand, which dissociates the newly formed double strand of DNA and returns the sequence of events to the start

Each cycle takes several minutes (or longer depending on the length of DNA to be replicated); the cycle is repeated 20 - 40 times to increase the DNA copies by 220 - 240.

Measurement of DNA

To measure the DNA fragments present, electrophoresis is performed. By applying an electric current to the DNA molecules while the are suspended in an agarose gel, they will move a particular distance based upon their molecular weight (ie. size of DNA strand). This can then be compared with a DNA ladder to analyse the size of the DNA molecules present. Because the number of copies of DNA has been amplified by at least 220 times, even a small amount of gene expression (through transcription of RNA) can be detected.

Alternative uses of DNA generated through RT-PCR

The DNA produced through RT-PCR can be inserted into cells (usually bacteria) that then causes them to express the gene and produce its protein product.

Use of RT-PCR in Oncology

RT-PCR is particularly useful to see whether a gene under investigation is being expressed by a malignant cell. It can also be used as an initial step for microarray testing if the expression of all the genes within a cell is under investigation.


Microarray Gene Profiling

Microarray gene profiling is used to examine the expression of genes in a cell, and compare it against other cell types. A microarray contains thousands of small probes that present a short strand of the genome, arranged in sequence (or 'spots').
Microarray Gene Profiling occurs in several steps:

  • mRNA from two different cells is removed from the nucleus
  • RT-PCR is performed to convert the RNA strands into complementary DNA strands
  • The DNA strands are labelled with a marker - often a protein which causes a colour change
  • The cDNA strands are applied to the microarray and hybridisation between the DNA strands in the solution and the DNA probes on the array occurs
  • The amount of binding that occurs is reflected by the colour change seen on the array
  • A computer then reads the intensity of the colour to determine the expression of particular genes

Use in Oncology

Microarrays are useful tools in exploring genes that might be involved in carcinogenesis. If a gene is significantly active in a cancer cell, and not active in the normal cell, it is possible that a mutation in that gene (or a gene that promotes its activation) is present.


References

Wikipedia (sorry)


Links

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