What is Polymerase Chain Reaction (PCR)?
A fast and inexpensive technique that exponentially amplifies target DNA.
Who invented PCR and when?
Kary Mullis in 1983.
For what invention did Kary Mullis receive the Nobel Prize in Chemistry?
For inventing PCR in 1993.
Name one application of PCR.
Genotyping.
What is required as the DNA polymerase in basic PCR?
Heat-resistant Taq DNA polymerase.
How many primers are needed in basic PCR?
Two primers with free 3'OH groups.
Which ions are usually used as bivalent cations in PCR buffer?
Magnesium chloride (MgCl2).
What machine is essential for performing PCR?
A PCR machine that controls heating and cooling cycles.
What are the three steps in each PCR cycle and their temperatures?
Denaturation at 95°C, annealing at 50–65°C, and extension at 72°C.
How many times are PCR cycles usually repeated?
20 to 40 times depending on starting copies.
How many copies can PCR produce from one or a few starting copies?
Billions of copies under efficient conditions.
How many double-stranded DNA molecules are produced in the first PCR cycle?
Two double-stranded DNA molecules.
How many double-stranded DNA molecules are produced in the second PCR cycle?
Four double-stranded DNA molecules.
How many double-stranded DNA molecules are produced in the third PCR cycle?
Eight double-stranded DNA molecules.
What is the melting temperature (Tm) of DNA?
The temperature where 50% of duplex DNA dissociates into single strands.
What does the Tm indicate about DNA duplex?
It indicates duplex stability.
How does PCR specificity relate to primer Tm?
PCR specificity depends strongly on primer Tm.
What is the common formula for primer melting temperature (Tm)?
What adjustment is made to Tm when setting up a PCR machine?
Subtract 5 degrees from the calculated Tm.
What are PCR primers?
Synthetic DNA oligonucleotides about 15 to 30 bases long.
Where do PCR primers bind?
To complementary sequences flanking the target region.
What is the recommended Tm range for PCR primers?
Between 55 and 70°C with no more than 5°C difference between primers.
What types of DNA can serve as template DNA in PCR?
Genomic DNA, complementary DNA, plasmid DNA, or a PCR product.
What is the typical starting amount of plasmid DNA in a 50 µL PCR?
0.1–1 ng of plasmid DNA.
What is the typical starting amount of genomic DNA in a 50 µL PCR?
5–50 ng of genomic DNA.
How does increasing template DNA amount affect PCR specificity?
It increases the risk of nonspecific amplification.
What is the half-life of Taq DNA polymerase at 95°C?
About 40 minutes.
At what rate does Taq polymerase incorporate nucleotides at 70°C?
About 60 bases per second.
What is the typical amount of DNA polymerase used in a 50 µL PCR?
1 to 2 units of DNA polymerase.
What pH range does PCR buffer usually have?
Between 8.0 and 9.5.
What is a plasmid vector designed to clone?
DNA fragments generally smaller than 10,000 base pairs in bacteria.
What essential elements do plasmid vectors contain?
At least one bacterial origin of replication and one antibiotic resistance gene.
What does the plasmid origin of replication enable?
Plasmids to replicate independently of bacterial chromosome replication.
What happens when the corresponding antibiotic is applied to bacterial cultures?
Bacteria without the plasmid are killed.
How long is the Bluescript pBSKSII+ plasmid?
2961 base pairs.
Which antibiotic resistance gene does the Bluescript pBSKSII+ plasmid contain?
An ampicillin-resistance gene.
What origin of replication does the Bluescript pBSKSII+ plasmid have?
A ColE1 origin of replication.
What special feature do the multiple cloning sites of Bluescript pBSKSII+ plasmid have?
They contain unique restriction sites.
What does a restriction enzyme recognize and do to DNA?
It recognizes a specific short DNA sequence and cleaves DNA at that site.
What are the possible lengths of restriction enzyme recognition sites?
They can be 4, 6, 8, 10, or 12 base pairs long.
Name three types of ends generated by restriction digestion.
5' cohesive ends, blunt ends, and 3' cohesive ends.
What type of ends does EcoRI generate after cutting?
5' cohesive ends.
Why is NotI's recognition site rarer than EcoRI's?
Because NotI recognizes an 8-base-pair palindromic sequence, longer than EcoRI's 6-base-pair site.
What is the purpose of molecular cloning?
To produce recombinant DNA molecules that replicate in a host organism.
What must DNA fragment and plasmid have before insertion in cloning?
Compatible ends to form phosphodiester bonds.
Test your knowledge with 20 questions on PCR and Plasmid Cloning.
1. What is the primary purpose of the Polymerase Chain Reaction?
2. Which combination contains the essential components of a basic PCR reaction?
Review the complete course in the study sheet for PCR and Plasmid Cloning.
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