Question: A gene containing 3 exons (A, B and C) and its corresponding cDNA are analyzed by PCR and DNA/DNA…

A gene containing 3 exons (A, B and C) and its corresponding
cDNA are analyzed by PCR and
DNA/DNA hybridization followed by nuclease digestion. The exon
order in the gene is -A-B-C-, and
previous experiments demonstrate that B is at an equal distance
from A and C. Thus, introns 1 and 2
are the same size.

1) The sense primer P1, anneals to the beginning of exon A, and
the antisense primer P2, anneals to
the end of exon C. P1 and P2 are used to PCR amplify DNA out of a
prepared cDNA library. Despite
the high specificity of both primers, the PCR reaction produces two
products with the following sizes:
Product B1: 750 bp Product B2: 350 bp.
Explain this result and calculate the length of exon B knowing that
the length of exons A and C are 150
bp and 200 bp respectively (3 pts).

2) In the following hybridization experiments, radioactive
single-stranded genomic DNA
encompassing the region coding for A, B and C is mixed with
unlabeled single-stranded DNA derived
from PCR Product B2. The resulting DNA hybrids are digested either
by S1 nuclease or exonuclease
VII. Both enzymes digest single-stranded nucleic acids, but only S1
nuclease is both an endonuclease
(does not need a free 3’ or 5’ end to initiate the cleavage of
single stranded DNA) and an exonuclease
(needs to start the cleavage of single stranded DNA by a 3’ or 5’
free end).
The products of both nuclease digestions are then resolved by gel
electrophoresis in neutral
(maintaining double-stranded DNA structure) or alkaline (inducing
the dissociation of DNA strands)
conditions. Only the radioactive DNA is visualized by
autoradiography (see below), and the size of
the fragments is calculated. Briefly explain the results obtained
and calculate the size of the introns.
(4 pts)