Assignment: In a genetic department, PCR wishes to amplify an area in gene Q to

Question

Assignment:

In a genetic department, PCR wishes to amplify an area in gene Q to investigate whether it contains a mutation.

Below is a sample of gene Q's coding string. The regions where PCR primers can be placed on either side of the mutation are marked with gray. The sequence between the primers is not specified, but it is 50 bp long.

1. Identify the sequences of the two primers used for the PCR analysis. Enter the 5 'and 3' ends of the primer.

2.Read the size of the PCR fragment formed by a successful PCR reaction. Show your calculation.

3. Specify a technique that can be used to determine if the PCR fragment has the expected size. Describe the principle behind the technique.

4. Specify a technique that can be used to determine if gene Q is mutated in the area between the PCR primers.

PCR is made with the designed primer, which is assumed to be constructed correctly. The following three temperatures are exchanged during the cycling process: 95 ° C, 65 ° C, 72 ° C.

5. Describe briefly what is expected to happen at each of the three temperatures.

However, it appears that the PCR reaction has not led to the formation of a product.

6. What temperature will you change and how to make the PCR reaction work? Justify your answer.

Explanation / Answer

1. forward primer-5’GTATTACTTCAAATG 3‘

   reverse primer- 3' GAATTACGTTTAAGA 5'

2. as the forward and reverse primer each contains 15 nucleotdies each, altogether 30 nucleotides / bp.

thus total PCR product size = 30 +50bp=80 bp

3. Gel electrophoresis is the technique which can be used to see whether the we have the PCR product at expected size or not. DNA fragments are separated according to size in gel electrophoresis.

4. Hybridization is a technique which can be used to see whther the area betwe the primers is mutated or not.

5. 95 oC- The double bonded DNA template is denatured.

    65oC- primers will anneal or added to the the strands

     72 oC- new DNA strands are synthesized.

6. I will change the annealing tempertue, as this is the temperature where primers are attached to the single strnded DNA. Thus changing this temperature will have effect on the efficiency of primer binding and PCR synthsis.

Related Questions

Navigate

• Browse (All)
• Browse A
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.