Question

You are using PCR to amplify a 300 bp target sequence, a portion of Gene X, from human genomic DNA isolated from patients' blood samples. The instructions for this procedure tell you to include Samples A and B, whose contents are listed below, with each batch of patient samples that you run.

Ingredients	Sample A	Sample B
10x PCR Buffer (Tris,KCI,MgCl2,BSA)	5 mL	5 mL
H2O	37.8mL	38.8mL
dNTP's	3 mL	3 mL
Taq DNA polymerase	0.2 mL	0.2 mL
Primers	3 mL	3 ml
Purified Gene X	1 ml	none

a) In one sentence or less, what is the purpose of Sample A?
b) In one sentence or less, what is the purpose of Sample B?
c) After the PCR amplification, an aliquot of each sample is electrophoresed on an agarose gel and visualized using ethidium bromide. The bands seen on the gels from three separate experiments are described below. Briefly describe what these results indicate.
Experiment 1: Both Samples A and B have a 300 bp band.
Experiment 2: Sample A has a 300 bp band; Sample B has no bands.
Experiment 3: Neither Sample A nor Sample B have bands.

For each unintended result (experimental problem) listed below:
- state ONE probable cause of the problem

- describe why this would produce the unintended result

a. After electrophoresing the products of your PCR reaction, you saw 3-5 bands in each sample. You have previous data to indicate that only one band should have been present.

a) Describe the essential components of a polymerase chain reaction.

b) Give 4 mechanisms by which the reaction may be inhibited and cause falsely negative results.

Answer 1

answering in order of questions been asked:

A) sample A will serve as a reference for presence of gene X. (it will act as control for reaction.)

B) sample B is from patient will serve as a test sample, i.e. we will checking the presence of gene X in this sample.

C) 1) experiment 1 indicates that PCR has worked well and it can be confirmed that the patient has presence of gene X.

2) experiment 2 indicates that PCR has worked well since we can see band in sample A, but absence of band in   sample B indicates the absence of gene X in patient.

3) experiment 3 indicates that the PCR has not worked properly, since we could not see any bad in either of the sample.

D) If we are seeing the 3-5 bands in each of the samples then the most possible reason is that the primers have amplified non-specific regions. this means primers taken were not efficient.

reason = non-specific primers can bind with multiple regions and carry out the amplification for those regions leading to multiple band observation on gel. if problem would have been in sample the we should have seen multiple band only in case of sample B, since we have taken purified gene X template for sample A. so problem is with primer and therefore it is showing the multiple bands on gel.

E) component listed in the reaction mixture stated in the problem are the essential components of the reaction.

F) 4 mechanism by which the reaction may be inhibited and cause falsely negative results.

1) wrong template used for PCR reaction

2) wrong combination of primer used for reaction

3) non-specific primers used for reaction can cause false negative results.

4) difference in PCR condition can also lead to false negative results. e.g. difference in melting temperature of the primer pair, or poorly designed primers for GC content.