2. This question concerns PCR and DNA sequencing. a. Describe what would happen

Question

2. This question concerns PCR and DNA sequencing. a. Describe what would happen in a PCR experiment if you only used one primer instead of two primers. b. Suppose you perform PCR using only one copy of a double-stranded DNA molecule. How many copies of this double-stranded DNA molecule would be present after 15 cycles? c. We usually think of enzymes being the most active at around 37?C (near body temperature). In PCR, however, DNA synthesis occurs at 72?C during each cycle. What is unique about the DNA polymerase that is used in PCR? d. Dideoxynucleotides (ddNTPs) play an important role in DNA sequencing. What is the structural difference between a nucleotide (NTP), a deoxynucleotide (dNTP), and a dideoxynucleotide (ddNTP).

Explanation / Answer

2a. PCR is possible with a single primer. The single primer will bind to one strand of DNA and Taq polymerase will perform strand extension. Double the number of molecules will however not be obtained per cycle. Hence, the PCR amplification will be a linear rather than an exponential amplification. Hence, amount of product formed will be much reduced and it will take a long time for PCR to be completed.

b. During each replication cycle in PCR, the number of molecules of the target sequence doubles. This is due to products and templates of previous round of replication are the templates for the next round. Hence, n rounds of replication produces 2n number of target sequence, where n= number of PCR cycle.

After 15 PCR cycles, one double stranded DNA forms,

1* 215= 32768 molecules of Double stranded DNA.

c. Taq DNA polymerase is a thermostable DNA polymerase obtained from Thermus aquaticus. This polymerase is used in PCR as it can tolerate temperature of high temperatures of 940C, which is used to denature DNA in PCR. It is not inactivated at temperature. Its optimum enzyme activity is at 750C and hence, can be used for DNA synthesis at the temperature of 720C. Hence, Taq DNA polymerase is used for PCR.

d. Nucleotides or NTPs is a nucleotide triphosphates that is the building block of RNA. It contains a ribose sugar that has OH group at second carbon of the sugar moiety. Hence, it is a ribose sugar that is linked to a phosphate group and a nitrogen bases. NTPs have three phosphate groups, a ribose sugar and a nitrogen bases.

Deoxyribonucleotides or dNTPs are similar to NTPs with a sugar, three phosphates, and a nitrogenous base. However, they have deoxyribose sugar instead ribose sugar. The hydroxyl group (2’ OH) is deoxygenated and replaced by hydrogen atom at C2 of the sugar moiety. dNTPs are building blocks of DNA.

Dideoxyribonucleotides (ddNTPs) are used for Sanger Sequencing. They are similar to dNTPs except the 3’ OH group of deoxyribose sugar is deoxygenated and replaced by H. Hence, ddNTPs lack both 2’OH and 3’ OH of ribose sugar. Due to lack of 3’ OH group, the extension of DNA cannot occur and the chain is terminated.

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