PCR and DNA Profiling Techniques Review

Flashcards covering the functions, steps, and variations of PCR, as well as key DNA profiling methods.

What is the function of the Polymerase Chain Reaction (PCR)?

It is a rapid, inexpensive, and simple molecular technique used to amplify a specific region of DNA in vitro.

Who discovered PCR and in what year?

It was discovered in 1983 by the American biochemist, Kary Mullis.

What are the three key steps in a PCR cycle?

Denaturing, annealing, and extension (or elongation).

What is the purpose and typical temperature of the denaturing step in PCR?

Heating the mixture to 94-96 degrees Celsius to separate the double-stranded DNA.

What is the enzyme required for PCR and why is it special?

Thermus aquaticus polymerase (Taq polymerase), isolated from T. aquaticus bacteria that lives in hot springs, allowing it to tolerate and function well at the extreme high temperatures required for the reaction.

What are primers in PCR and what do they bind to during the annealing step?

Short, single-stranded nucleotide sequences (20-25 bp) that are complementary to the 3' ends of the sense and antisense strands, binding to the area just outside the target DNA region at 50-60 degrees Celsius.

What is reverse transcriptase PCR (RT-PCR) used for?

It is commonly used to study changes in cellular activity by isolating expressed RNA, generating complementary DNA (cDNA) using reverse transcriptase, and then running PCR amplification.

What is the major difference between one-step and two-step RT-PCR?

One-step RT-PCR performs cDNA synthesis from RNA and PCR amplification in a single tube, while two-step RT-PCR performs the two processes in separate tubes.

What is the primary function of nested PCR?

It is designed to increase the specificity of the amplified DNA by using two sets of primer pairs in two successive reactions (an outer primer pair followed by an inner primer pair).

How does real-time or quantitative PCR (qPCR) measure DNA quantity?

It measures the quantity of amplified DNA in real-time using fluorescently labeled DNA probes or fluorescent dyes that bind to the DNA as it amplifies. The fluorescence intensity is directly proportional to the amount of amplified DNA present.

Name the three main techniques used in DNA profiling or DNA fingerprinting.

Restriction Fragment Length Polymorphism (RFLP), Variable Number Tandem Repeats (VNTR), and Short Tandem Repeats (STR).

What is the main difference between Variable Number Tandem Repeats (VNTRs) and Short Tandem Repeats (STRs)?

VNTRs are minisatellites, usually 10-60 bp repeats, while STRs are microsatellites, usually 2-6 bp repeats.

Which DNA profiling technique requires the least amount of DNA and is the most recent and common method used?

Short Tandem Repeats (STRs).