1. Imagine you want to make a cDNA library starting from a sample of RNA purified from mouse liver cells.

(a) The first task is to make a cDNA copy of the mRNA population. I am assuming here that we are interested only in looking at mRNAs, which have polyA tails consisting of variable numbers of A residues, mainly in the range of 50-150. For first strand cDNA synthesis with reverse transcriptase you need to choose a primer.
You might choose an oligo-dT primer that is simply a run of 20 consecutive T residues.
You are (correctly) told that an alternative choice that can work is to use “random hexamers” (a mixture of all possible oligonucleotide sequences of 6nt length; 46 oligos).

(i) How are random hexamers most economically made?

(ii) How is it that they work to prime cDNA synthesis even though at 37C and low salt concentration (conditions suitable for reverse transcriptase) two sequences with complementary regions of only 6 bp are mostly single-stranded rather than hybridized?

(iii) Even though random hexamers can technically be used what are TWO clear advantages of using oligo-dT for making a useful cDNA library?(“Useful” means most clones in the library are good representations of mRNAs in the starting sample).

(b) After first strand synthesis, the RNA can be degraded. There are then several possible strategies for priming synthesis of the second cDNA strand. One possibility is to tail the first cDNA strand with a run of dC residues (a variable number that cannot be strictly controlled but perhaps mainly in the range of 20-40) using terminal transferase. Then you could use a primer that is oligo-dG (say, just a run of 15 G residues).

(i) If you used a (dT)20 primer for the first strand as suggested in (a), then tailed with dC residues and used a (dG)15 primer for the second strand, what would a typical ds cDNA product look like (easiest to draw it but be sure to indicate how many Ts, As, Cs and Gs are at each end either as an exact number or a range, and making clear whether ends are blunt or have single-stranded regions)? [1]

(ii) Could you have used a slightly different primer for first strand synthesis so that there would be exactly 20 T residues at one end of the molecule you have drawn above? Explain.

(iii) You add T4 DNA polymerase to the products you have represented in (i). What dNTPs should you add if you want to end up with cDNAs that have blunt-ends on both sides? Explain.

(iv) You add T4 DNA polymerase to the products you have represented in (i). What dNTPs should you add if you want to end up with cDNAs that are blunt at one end but have single-stranded oligo-dG overhangs at the other end? Explain.

(c) Imagine you have the product described in (iv) above and that you have separate preparations of the left arm and right arm of a lambda insertion vector, each with a blunt end at the cloning site. You want to produce recombinant phage that have a cDNA with the end corresponding to the 5’ end of the parent mRNA joined to the left arm and the other cDNA end joined to the right lambda arm.

(i) To accomplish this objective what steps would you take (in order) to manipulate the DNAs? Note there is more than one point available and so there are possibly several steps. Be sure to mention when T4 DNA ligase is added (and when it is not present).

1. Imagine you want to make a cDNA library starting from a sample o...