Obtaining full length cDNA - Obtaining full length cDNA using Xenopus (Oct/10/2005 )
Hello everyone. I'm new to these forums and this is my first post. I have been browsing these forums for a while, but now decided to post.
I need help with a coursework question, not answers, but just advice on a good place to start. My lecturer recently gave me the following question:
Please suggest a procedure for obtaining a full-length cDNA clone for the putative novel opsin expressed in Xenopus skin. You have access to the following material:
A colony of African clawed frogs (Xenopus laevis)
Cloned fragments of Xenopus rhodopsin and violet-sensitive cone opsin cDNA
Deep-frozen human retina
Standard Molecular Biology reagents
Those were his words on what we definetely have access to. However, he said we may utilise other things not mentioned e.g. PCR.
Using his notes and the reccommended textbooks, I came up with this answer
There are many ways to obtain a full-length cDNA clone. One method is via the utilisation of in situ hybridisation. By taking a section of tissue (preferably one that is in the process of cell division) from the Xenopus frog and mounting the tissue on a prepared slide, the cells of the organism’s tissue can be treated with a fixative (e.g. formaldehyde) to preserve the tissue before undergoing incubation with sodium hydroxide and ribonuclease. The purpose of this addition is to denature the molecules of DNA present in the tissue. The denaturation of DNA initiates the breakdown of base pairing between individual polynucleotide strands, causing the chromosomes found on the Xenopus tissue to "unpack" slightly. This structural change in the chromosome exposes particular segments of DNA that would normally be enclosed within the chromosomal body. A sample of the gene (the cloned fragments of Xenopus violet sensitive cone opsin cDNA), once labelled with a radioactive probe, is then applied to the slide (on which chromosome are present). This starts the process of hybridization between both gene and the chromosomal replica, which results in the formation of a dark spot on an autoradiograph. This spot denotes the position of the gene on the chromosome.
I somehow feel I havent answered the question, or am missing something, but have went through the notes and books time and time again. I would be very grateful for your help. The deadline for this isn't too close, but I thought it best to start early.
Thanx for any responses. I am reading a BSc (Hons) Molecular Biology degree is that helps.
Your problem is that your procedure as outlined thus far might get you a DNA (chromosomal) clone, but not a cDNA clone. A cDNA clone must be made from mRNA which has been translated into DNA by reverse transcriptase. In this case, we're going from RNA to DNA, backwards from what most people think of.
The reason for doing it this way is that, unlike bacterial genes, eukaryotic organsims have stretches of apparently non-functional DNA (introns) interspersed with the coding sequences (exons). The introns are spliced out before translation of the mRNA into protein, thus if we want the sequence of the gene, with all functional segments (exons) included and non-funtional segements (introns) excluded, we start from the mRNA.
You do not want to use ribonuclease, as that will destroy the RNA. At the first stage of the procedure, DNA is a contaminant.
See if this helps get you pointed in the right direction...
Full length cDNA means of a gene can be obtained by reverse transcribing the mRNA of that particular gene. There are several methods of obtaining a full length cDNA . RACE PCR is one example. The idea is to use a technique that utilizes mRNA of that gene to make cDNA without interrupting the reverse transcriptase. Once the full length cDNA is obtained by RACE PCR it can be cloned into a plasmid.
Hope that helps. Refer to ambion website for more help
Thank you both very much, you both have really helped me a lot. Just wondering, what is Ambion?
its the name of a company..above is the weblink