What would happen if you mistakenly added some of the Instagene matrix to your P

Question

What would happen if you mistakenly added some of the Instagene matrix to your PCR reaction tube?

Why is it necessary to have a primer on each side of the DNA segment to be amplified?

Describe the 3 main steps of each cycle of PCR amplification and what reactions occur at each temperature.

The first cycle does not yield the precise length target DNA sequence (the first cycle yields larger

fragments). Precise length double-stranded DNA is not formed until cycle 3. Why? (it may help to draw it out)

List all of the needed components of PCR and what the function is of each.

Explanation / Answer

1. Instagene matrix is a solution that contains negatively charged microscopic beads that would bind to the negatively charged cations for example magnesium. When the instagene matrix is added to the PCR mixture, there will be binding of the Mg++ ions that would inhibit the activity of any DNAse present in the solution, by mistake, and hence, the integrity of the genomic DNA would be maintained.

2. While performing the polymerase chain reaction, there will be addition of primer on either end of the DNA segment that is supposed to be amplified. Primers are short stretches of DNA 18-40 bp in length, that is complementary to the DNA which is going to be amplified. One of the primer attaches to the 5' end of the DNA sequence and the second primer hybridises onto the 3' end of the DNA sequence to be amplified. Hence, it is required to have two primers attached on both strands of the target sequence.

3. The main steps in PCR are :

----> Denaturation: In this step, melting of the double stranded DNA occurs. Here, the reaction occurs at 94–98 °C and the reaction is carried out for 20-30 seconds. This results in melting of the DNA and thehydrogen bonds holding the complementary bases are broken.

----> Annealing : In the annealing step, there is a lower temperature of the mixture allowing the primers to attach to the single stranded denatured DNA. The temperature of this reaction is 50–65 °C and the reaction is carried out for 20-40 seconds. In this step, the temperature has to be kept such that the primers bind perfectly on the region complementary to the strand. When the primer sequence attaches perfectly on to the complementary part of the DNA, and thus there is formation of of hydrogen bonding between primer and target DNA.

----> Elongation : In the elongation step, the temperature of the reaction mixture in this step is 75–80 °C with Taq polymerase enzyme used to synthesise new DNA strands which are complementary to the template strand. In this step, new free dNTPs are added in order to continue the synthesis. In order to obtain the appropriate number of copies of the DNA, multiple cycles are required of elongation.

In the final step, the reaction chamber is cooled to 4–15 °C for storage of short term products.

5. Components of PCR and their functions :

a) DNA template : containing target sequence

b) DNA polymerase: used to synthesise new strands

c) Primers : short stretches that attach to target sequence on the two denatured strands ( Both forward and reverse primers ).

d) dNTPs : used in the elongation step to add more nucleotides to the growing strand.

e) reaction buffer : to maintain a stable pH. Contains MgCl2 which acts as co factor for Taq polymerase.

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