Molecular Biology Lecture Review (recording)

Flashcards designed to review key concepts from a lecture on molecular biology, focusing on PCR, primer design, and cloning techniques.

What is the probability of finding a complementary sequence in template DNA if the primer length is 6 bases?

Approximately 1 out of every 4,096 bases.

What happens if a primer is too short during DNA amplification?

It increases the chance that the primer will bind to a complementary sequence randomly.

What is TM in the context of DNA?

TM refers to the melting temperature at which 50% of the DNA is single-stranded and 50% is double-stranded.

Why is it important to know the melting temperature (TM) of primers?

It helps to determine the annealing temperature for PCR, which is typically 2-4 degrees Celsius below the TM of the primers.

What are primer dimers and why are they a problem in PCR?

Primer dimers occur when primers bind to each other instead of the target DNA, leading to non-specific amplification.

How does a longer primer affect the binding to the template DNA?

The longer the primer, the less likely it is to bind to non-specific sites due to greater specificity.

What are degenerate primers?

Primers designed based on amino acid sequences that account for multiple codons that encode the same amino acid.

What is the significance of using Taq DNA polymerase in PCR?

Taq polymerase is heat-stable, allowing for efficient DNA synthesis during the denaturation step of PCR.

What is a key disadvantage of Taq DNA polymerase?

It lacks proofreading ability, which can lead to higher error rates in PCR products.

What is the process and purpose of TA cloning?

TA cloning involves adding an A overhang to PCR products that can anneal to T overhangs on plasmids for easier insertion.

What consequences can mutations in PCR products introduced by DNA polymerases have?

Mutations can result in non-functional or incorrectly functioning proteins, which could affect downstream applications.

How do you ensure you have a plasmid with no errors after amplification?

By sequencing the insert of the plasmid to check for errors before using it for further applications.

What is the melting temperature (TM)?

The temperature at which 50% of the DNA strands are single-stranded and 50% are double-stranded.

Why should primers be 18-30 bases long?

Longer primers decrease the likelihood of non-specific binding and increase specificity in hybridization.

How does temperature affect the hybridization of DNA during PCR?

Higher temperatures can lead to complete denaturation, while lower temperatures may allow for mismatches.

What are intramolecular base pairings in primers?

Base pairings that occur within a single primer, potentially reducing its ability to bind effectively to template DNA.

What is the typical error rate for Taq DNA polymerase?

About 1 in every 5,000 base pairs.

What can you do if the sequence of the gene you want to amplify is unknown?

Design consensus primers based on sequences known from related organisms.

What is the role of topoisomerase in TOPO cloning?

It catalyzes the formation of covalent bonds between the insert and the plasmid without the need for an external ligase.