MOLBIO LAB 8

What is the primary purpose of performing a Polymerase Chain Reaction (PCR) after nucleic acid extraction?

To amplify the nucleic acid so it is suitable for further testing

Which component of the PCR master mix serves as the template providing the order of nucleotide bases?

Double-stranded DNA

What are the four specific deoxynucleotide triphosphates (dNTPs) included in a PCR master mix?

dATP,dGTP,dCTP, and dTTP deoxyadenosine triphosphate deoxyguanosine triphosphate deoxycytosine triphosphate deoxythymidine triphosphate

What is the role of DNA polymerase in a PCR reaction?

It catalyzes the addition of nucleotides to the growing DNA strand

To which specific component of the master mix does DNA polymerase add subsequent bases?

Primer

To which ends of the DNA target are primers designed to bind?

The 3′ and 5′ends

What is the practical length range for primers used in PCR?

20−30 base pairs

Why must the Guanine and Cytosine (GC) content of a primer be limited to approximately 50%?

High GC content forms stable imperfect hybrids, while low GC content depresses the melting temperature

What is the purpose of ensuring the 3′end of a primer is GC-rich?

To enhance the annealing of the end that will be extended by the polymerase

In primer design, how many Guanines or Cytosines should be avoided in the last 5 bases of the 3' end to prevent non-specific priming?

More than 3

What is the maximum allowable difference in melting temperature (Tm) between forward and reverse primers?

5 degrees Celsius

Formula: What is the formula used to calculate the melting temperature (Tm) of a primer?

Tm =4(G+C)+2(A+T)

Under what condition do primer dimers typically occur?

When there are three or more complementary bases at the 3′ ends of the primers.

Why is the formation of primer dimers detrimental to a PCR reaction?

They compete for primers and reagents, inhibiting the amplification of the intended DNA target

To which end of a primer can unrelated sequences, such as fluorescent labels or biotin, be added?

The 5′ end

Concept: Primer Melting Temperature (Tm) Definition: The temperature at which one half of the DNA duplex dissociates into single strands.

Primer Melting Temperature (Tm)

What is the ideal melting temperature (Tm) range for PCR primers to achieve the best results?

52 C−58 C

What secondary structure is formed by internal folds caused by base-pairing within a single primer molecule?

Hairpin formation

What is the potential byproduct of PCR that is bigger than ordinary primers because they consist of 2 primary molecules?

Primer dimers

From which organism is the most commonly used Taq DNA Polymerase isolated?

Thermus aquaticus

What is the ideal working temperature for Taq DNA Polymerase?

72 C

What is the half-life of Taq DNA Polymerase at 95 C?

40 minutes

What is the standard concentration range of Taq polymerase per 100 μL reaction volume?

1−2.5 units

Name two other thermostable DNA polymerases besides Taq used in PCR

Pwo and Tth DNA polymerases

Tth DNA polymerase

Thermus thermophilus

Pwo DNA polymerase

Pyrococcus woesei

What is the typical concentration of each dNTP in the final reaction?

200 μM

What is the typical pH range for a PCR buffer?

8.0 to 9.5

What is the function of Reverse Transcriptase in RT-PCR?

It creates a complementary DNA copy from an RNA template

Name three viruses with RNA genomes that are commonly detected using RT-PCR

HIV, HCV, COVID-19, or Influenza

Lyophilized

A substance, such as a primer, that is in a powdered form

How is the volume of ultrapure water calculated to resuspend a lyophilized primer to a 100 μMstock solution?

Multiply the oligo yield in nmol by 10

What is the standard concentration for a 'working solution' of primers in the lab protocol?

10 μM

How is the 'pipetting error' factor determined if the total number of samples and controls is between 1-14?

A pipetting error of 1 is added

What is the pipetting error factor added if the total number of samples and controls is >15?

A pipetting error of 2 is added

What is the total volume of master mix required for a single PCR test according to the provided laboratory table?

23 μL

The forward primer for the 16s rRNA is ____.

21 base pairs

The reverse primer for the 16s rRNA is ____.

19 base pairs

What are the components found in the PCR master mix?

Primers; DNA polymerase; Deoxynucleotide triphosphate; PCR buffer; Magnesium chloride

It functions as a cofactor of the DNA polymerase by enabling incorporations if the nucleotide of the dNTPs during polymerization

Magnesium ions

What are the components of the PCR buffer?

Tris-HCl; Ammonium sulfate; Magnesium chloride (sometimes)