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mRNA and cDNA cloning

(i) If you have a full-length cDNA clone that is a perfect copy of an mRNA species you ought to be able to predict the primary amino acid sequence of a translation product. For a mouse gene how would you do that?
(ii) If only one species of mRNA were produced for the gene in question how would you determine that the cDNA you examine is (or is not) most likely a full-length copy of the mRNA? Give TWO criteria you could use (WITH EXPLANATIONS). The criteria could involve existing information or performing additional experiments.

(iii) If more than one mRNA can be produced for a certain gene you might find that you can clone two types of cDNA from, say, liver RNA. These cDNA sequences might show that one form splices exon 1 to exon 2, and exon 2 to exon3 while the other might splice exon 1 straight to exon 3. How could you most easily determine (experimentally) if the same two splice forms exist in lymphocyte mRNA?

(iv) Normalizing cDNA libraries can help to collect representatives of different genes more economically by avoiding picking up many cDNA copies of an abundant mRNA for every copy of a less well expressed mRNA. Please explain the main principles involved in normalization (not the experimental details or any other part of cDNA library construction that is irrelevant.

(v) Does normalization also help you to collect differently spliced forms of a given gene by equalizing the relative abundance of those differently spliced mRNAs? And if it does why?

Solution Preview

(i) With the mouse genome sequenced, you can easily predict the primary amino acid sequence of a translation product. This can be done by using the "blastx", which is searching the protein database using a translated nucleotide sequence. Following is the link:
http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastx&BLAST_PROGRAMS=blastx&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome

In the "enter the query sequence" you enter the sequence you have and then chose the organism as mouse. After blasting the sequence with the mouse genome, you would get the protein and then click on the sequence of the protein to see the amino acids.

(ii) First of all the cDNA can be cloned and then sequenced. Since, the mouse genome is ...

Solution Summary

The mRNA and cDNA cloning issues are embedded.

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