Question

Which of the following answer(s) are true (select all that apply)?

a)PCR results in exclusively the desired region to be amplified.

b) PCR primers must be outside the region of interest.

c) PCR primers must be flanking the region of interest.

d) Sequencing primers must be flanking the region of interest.

e) All of the nucleotides used in Sanger Sequencing would also be able to be used for a PCR reaction where you are trying to amplify a gene of interest.

f) Sanger sequencing requires dNTPs

g) A standard PCR reaction requires ddNTPs Small DNA fragments on a gel would be closer to the wells on a gel than larger DNA fragments.

Answer 1

answer to the above question:

all the true statements are :

a) PCR results in exclusively the desired region to be amplified. because , specific forward and reverse primers are used for the fragment of interest, thus only the desired region will be amplified in all the cycles.

c)PCR primers must be flanking the region of interest.because in that case, only the desired segment of gene will be amplified , and no sequence information will be lost during amplification. if primers will be outside the region of interest , as said in (b) part , it will result in non specific amplification.

d) sequencing primers must be flanking the region of interest.in this case since we are using primers for sequencing that means its sequencing by synthesis , that means sequencing will begin after the primer ends , thus it(primer) must be flanking region of interest.

f) sanger sequencing requires dNTPs. the statement holds true because in sanger sequencing method along with ddNTPS , dNTPs are also required , in a specific ratio but if the statment would have said that it only requires dNTPS , then it must be wrong statement, but for now it is true.

statements which are false:

b) PCR primers must be outside region of interest. it is false beacuse, primers anywhere outside the region of interest is not prefered rather should be close enough or flanking the region of interest otherwise it will result in non specific amplification.

e)all the nucleotides used in sanger sequencing can be used in PCR where you are trying to amplify the gene of interest.the statement is wrong because in sanger sequencing ddNTPs are also used, which do not have 3' OH and thus will stop further elongation , which is not suited for a PCR reaction , set to amplify a gene of interest because in that case incorporation of a ddNTP will stop further amplification.

g) a standard pcr require ddNTPS, AND small fragments of DNA are near the well. both the statements individually are wrong because a PCR reaction for standard amplification does not requires ddNTPs , and smaller fragments on a gel run faster than bigger DNA fragments , so bigger fragments are closer to the wells rather than smaller DNA fragments.