You are given three primer sets by which to perform PCR with an unidentified bac

Question

You are given three primer sets by which to perform PCR with an unidentified bacterium pure culture sample. Primer set A: 1492R & F27 Primer set B: F343 & 534 Primer set C: R1492 & F519 Identify the expected length of each product for each of the given primer sets ( pt each). Primer set A: 1492R & F27-Product length- Primer set B: F343 & 534R-Product length: Primer set C: R1492 & F519-Product length Which primer set gives you largest amplicon (pt)? What does a larger amplicon mean for bacterial identification through the molecular method of identification (this means that you will need to think about the entire process from PCR through BLAST'ing, what does a larger amplicon mean, versus a shorter amplicon, in terms of your ultimate ability to identify an unknown bacterium)? (2 point)

Explanation / Answer

Expected product for the given primers:

So themaximum size of the amplicon we will get as a product will be 1492-27 = 1365 bp

2. F343 And R 534: Calculation similar to 1. so max amplicon product = 534-343 = 191bp

3. R1492 and F519: 1492-519 = 873bp

b. The primer set F27 and R1492 will give the maximum size of amplicon.

C. For identification purpose the ampliconn size should not be much larger, as it may demolish in between as well as there will be chance of false primer addition while amplification. Secondly when there will be sequencing, the sequencer do not read a much larger fraction, so having smaller amplicon are preferred for accurate and feasible results. Even if we have to amplify larger product we can use more than one primer producing smaller overlapping sequences, that can be read and a proper contig can be prepared.

D. In a gel electrophoresis the larger products of PCR move slowly and appear in top of the gel, and the smaller one moves faster and found at lower part.

So the lane A has the longest amplicon size so it was amplified with the first set of primer

Lane B is showing the smallest amplicon so primer 3 was used.

Lane C product was amplified with primer set 2.

E. After the PCR BLAST result we can identify the organism from genus to species as well as strain level depending upon the no. of sequence in the query subject. So if the sequences are larger in no. it gets more nearer similarity with the available database.

So if the query length is near to 1500 bp, it shows the result upto strain level, lesser will show upto species level and similarly the lowest will give idea about genus only.

So, E. Coli 01H57 will be identified by the sequence obtained by the largest product which is a result of amplification by primer set 1.

E.Coli will be obtained by primer set 2.

and the Escherichia genus will be identified by Primer set 3.

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