PCR Applications and Processes

These flashcards cover key concepts related to PCR, its applications, components, techniques, and implications in cloning and forensic science.

What are the main components of a PCR reaction mix?

Primers, deoxynucleotide triphosphates (dNTPs), buffer, and DNA polymerase.

What role do primates play in PCR?

Primers are short sequences of nucleotides that provide a starting point for DNA synthesis.

What is the purpose of the buffer in a PCR reaction?

To maintain a stable pH environment during the reaction.

What is the significance of fidelity in DNA polymerases?

Fidelity refers to the accuracy of DNA synthesis; higher fidelity means fewer errors in replication.

Name a property that some advanced DNA polymerases have that Taq polymerase does not.

Proofreading activity.

How does Taq polymerase differ from other DNA polymerases in terms of product?

Taq polymerase adds A overhangs to PCR products.

What is TA cloning in DNA cloning?

A method that utilizes A overhangs from PCR products to ligate into T overhangs on linearized plasmids.

What is the advantage of using topoisomerase over DNA ligase in cloning?

Topoisomerase can covalently bind and seal plasmids faster because it does not require additional components to bind to the plasmid.

What are degenerative primers?

Primers designed based on polypeptide sequences to amplify genes of interest, accounting for potential sequence variation.

What is nested PCR?

A technique where two sets of primers are used to increase specificity and yield of the target amplification.

What problem can arise from using Taq polymerase?

It can introduce errors during amplification due to its lack of proofreading ability.

How can one confirm successful cloning of a PCR product?

By performing colony PCR using insert-specific primers.

What does it mean when it's said that not all cells from a colony are clones of each other?

Cells from different colonies can have different genetic compositions.

What are positive and negative controls in PCR?

Positive controls confirm the PCR process works, while negative controls show that under false conditions, no amplification occurs.

How can PCR be used in forensic science?

PCR can amplify DNA from samples to identify individuals based on unique genetic markers.

What is the role of the enzyme beta-lactamase in the context of cloning?

Beta-lactamase confers resistance to ampicillin, allowing the selection of recombinant plasmids in bacteria.

What is a suicide vector?

A type of plasmid that leads to the death of bacteria unless they contain a recombinant insert that deactivates the toxic gene.

What is whole genome sequencing, and why is it significant?

Whole genome sequencing determines the complete DNA sequence of an organism's genome, which can aid in diagnostics and research.

Why might researchers prefer Taq polymerase despite its error-prone nature?

Taq polymerase is faster and less expensive compared to more accurate alternatives.