Urgent Help needed with this question. PCR theory

[ilovescience]

Please please can someone help me work out this question, I have an exam very soon, and I just don't understand it.

Below is a 320 base pair section of part of the human mitochondrial genome sequence

1 AACCTGACTA GAAAAGCTAT TACCTAAAAC AATTTCACAG
41 CACCAAATCT CCACCTCCAT CATCACCTCA ACCCAAAAAG
81 GCATAATTAA ACTTTACTTC CTCTCTTTCT TCTTCCCACT
121 CATCCTAACC CTACTCCTAA TCACATAACC TATTCCCCCG
161 AGCAATCTCA ATTACAATAT ATACACCAAC AAACAATGTT
201 CAACCAGTAA CTACTACTAA TCAACGCCCA TAATCATACA
241 AAGCCCCCGC ACCAATAGGA TCCTCCCGAA TCAACCCTGA
281 CCCCTCTCCT TCATAAATTA TTCAGCTTCC TACACTATTA


a) Three of the following PCR primers are forward primers and three are reverse primers for the above DNA sequence. Using various combinations of these primers, a total of 6 different PCR products can be produced. List the paired combinations of primers that would produce a PCR product from the above sequence. What would the size of those PCR products be?

Primer 1: 5’AAGCTATTACCTAAAA 3’
Primer 2: 5’TAATAGTGTAGGAAG 3’
Primer 3: 5’TTTGTATGATTATGGG 3’
Primer 4: 5’TAACCTATTCCCCCG 3’
Primer 5: 5’GTGATTAGGAGTAGGG 3’
Primer 6: 5’CCCCGCACCAATAGGAT 3’

b) The optimum annealing temperature for a primer can be estimated by multiplying the numbers of A’s and T’s by two and the number of G’s and C’s by four, then taking a total. It is desirable for both primers in a PCR you have similar annealing temperatures. With this in min, design a PCR primer, that when paired with primer 2, will amplify the whole 320 base pair section of sequence shown above.

c) You wish to amplify the whole 320 base pair section of sequence shown above and then to clone it into the EcoRI cloning site of pBIOL2004. The EcoRI recognition site is ‘GAATTC’. How might you modify the primers from (b) to facilitate cloning?

d) In the first few rounds of a PCR, the polymerase produces some newly –synthesised DNA strands that are longer that the distance between the 5’end s of the PCR primers. However, in later rounds, the proportion of newly synthesised DNA strands that are longer than the distance between the 5’ ends of the PCR primers becomes vanishingly small. Why is this ?


THANK YOU

[ilovescience]

so far, for part a) I got :
primer 1 + primer 2 = 333
primer 1 + primer 3 = 227
primer 1 + primer 5 = 131

primer 4 + primer 2 =175
primer 4 + primer 3 = 97
primer 4 + primer 5 =32

primer 6 + primer 2 = 76
primer 6 + primer 3 = 35
primer 6 + primer 5 = 133

However, these are the possible nine combinations and the question asks for SIX. which do I elminate?

[ilovescience]

If not, does anyone have a question with similar layout to it that they could post on here, and I can apply it as a worked example perhaps? thank you