Question

How would the yield of a PCR reaction be affected by the following mistakes?

Explain your answer.

1. You forgot to add template DNA

2. you added TWICE as much template DNA as is called for by the intructions

3. You forgot to add Taq polymerase

4. You add HALF as much Taq polymerase as is called for the by instructions

Answer 1

Q) How would the yield of a PCR reaction be affected by the following mistakes?

1. You forgot to add template DNA.

Answer:-

If we forgot to add DNA template, than we will not be able to get the genotype, because there will be no DNA product on which GEL electrophoresis can run.and DNA can not be amplified in absence of it, as this is the primary ingredient of PCR on which primer and taqpolymerase works.

Explanation:- PCR is a technique used to make many copies of a specific DNA region in a test tube (invivo), This DNA region can be  a gene whose function a researcher wants to understand, or a genetic marker used by forensic scientists to match crime scene DNA with suspects., DNA amplified by PCR may be sent for sequencing, and seen by gel electrophoresis, or cloned into a plasmid for other experiments. The ingredients or components of PCR are  Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks).

DNA template is the particular DNA sequence which we want to copy, so if we forgot to add Template DNA there will be no DNA on which the primer and taq polymerase can act and as a result PCR will stop .

Q) Case -2 - you added TWICE as much template DNA as is called for by the intructions.

Answer :- If Twice template DNA is added PCR is inhibited as there will not be proper denaturation of DNA.

Explanation:- In PCR denaturation of double stranded DNA is done at a particular temperature , so if there will be more template DNA strands than the given heat will not be sufficent to denature DNA, also the other ingredients will not be sufficient to proceede the steps of PCR.

Q ) Case-3- You forgot to add Taq polymerase

Answer:- Than primer will not be able to form complementary strand of DNA, DNA will not be amplified and PCR will come to a halt.

Explanation:- In PCR Taq polymerase is used because it is a heat stable enzyme, it can resist high heat used for Denaturation process to make DNA template in PCR, Taq polymerase plays a crucial role in building the complementary strand. It gets attached to the primer and keep on adding DNA bases to the single strand one-by-one in the 5’ to 3’ direction. so in absence of it there will be no amplification of DNA.

Q ) Case -4 You add HALF as much Taq polymerase as is called for the by instructions.

Answer:- If Taq polymerase is low than all PCR products will not be amplified.

Explanation:- As taq polymerase helps in attaching nucleotides to the primer, and helps in copying the DNA.so if concentration of taq polymerase will be low nucleotides attachment will be low to primer and all DNA can not be copied in PCR process.