Question

A single strand of DNA, 24 nucleotides long, with the sequence

5'-TTTCCCgggAAAgggTTTAAAggg-3'

is in a test tube. (Note that G's are shown in lowercase, so that your eye can better distinguish them from C's)

Other than the appropriate buffer solution, what else needs to go in the test tube to so that we end up with a piece of double stranded DNA, 24 base pairs long, with the above sequence comprising one of the two strands? (SELECT ALL THAT APPLY)

The primer 5'-TTTCCCg-3'

The primer 5'-CCCTTTA-3'

deoxynucleotide triphosphates

deoxynucleotide monophosphates

The primer 5'-CgggAAA-3'

The primer 5'-CCTTTCC-3'

The primer 5'-TAAAggg-3'

DNA unwindase

DNA polymerase

RNA polymerase

Answer 1

We have been given a single strand of DNA, 24 nucleotides long, with the sequence:

5'-TTTCCCgggAAAgggTTTAAAggg-3'

From this given single-stranded DNA (ssDNA), we need to synthesize a double stranded DNA (dsDNA), which is also 24 base pairs long, and with the given sequence comprising one of the two strands.

The given ssDNA would serve as a template strand for the synthesis of a complementary strand to form a double-stranded (ds) DNA. The reagents that need to go in the test tube other than the appropriate buffer solution and the given template, are the below three:

The primer 5'-CCCTTTA-3' - The 5' end of this primer would attach to the 3' end of the template strand perfectly as this sequence is complementary to the 3' end of the template strand in this manner:

5'-TTTCCCgggAAAgggTTTAAAggg-3'

3'-ATTTCCC-5'

All other given primers are not complementary to the 3' end of the template strand.

Also, the enzyme that synthesizes DNA, DNA polymerase, can only add nucleotides to an already existing strand or primer of DNA that is base paired with the template, therefore a primer is required for initiation of DNA synthesis.

deoxynucleotide triphosphates - Four deoxyribonucleotide triphosphates (dNTP's) are required for DNA synthesis. These are dATP, dGTP, dTTP and dCTP. The high energy phosphate bond between the alpha and beta phosphates is cleaved and the deoxynucleotide monophosphate is incorporated into the new DNA strand. Note that only dNTPs, but not deoxynucleotide monophosphates (dNMPs), can base pair with the bases in the template.    

DNA polymerase - DNA polymerase is required for the covalent joining of the incoming nucleotide to the primer, and it extends the newly synthesized strand in the 5' to 3' direction.

THESE SHOULD NOT BE ADDED TO THE TESTTUBE:

The other given primers 5'-TTTCCCg-3', 5'-CgggAAA-3', 5'-CCTTTCC-3', and 5'-TAAAggg-3' are not complementary to the 3' end of the template strand, and would not bind to it, so they should not be added.

DNA unwindase (or helicase) is essential during DNA replication because it unwinds or separates double-stranded DNA into single strands allowing each strand to be copied. Since here, DNA is synthesized from single stranded DNA, so no unwinding is necessary.

RNA polymerase is an enzyme that is responsible for copying a DNA sequence into an RNA sequence, duyring the process of transcription. Thus, it should not be added here.

deoxynucleotide monophosphates - As mentioned earlier, only dNTPs, but not deoxynucleotide monophosphates (dNMPs), can base pair with the nucleotides in the template, .The incoming dNTP first forms a base pair with a base in the template. Only then the DNA polymerase links the incoming base with the predecessor nucleotide on the primer. The high energy phosphate bond between the alpha and beta phosphates in dNTP is cleaved and the deoxynucleotide monophosphate is incorporated into the new DNA strand.