Hot Start PCR Principle: Steps and Techniques Quiz and Flashcards

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Questions and Answers

What happens to the sequestered component when the reaction temperature is high enough to melt the wax or agarose?

The sequestered component is released into the reaction mix.

What is the purpose of the precipitate in the chelation method?

The precipitate chelates magnesium ions from the reaction mixture and releases them at a reaction temperature between 50 and 95 ℃.

What is the purpose of chemical modification of the polymerase in the hot start PCR method?

The polymerase is reversibly inactivated by chemical modification.

What is the problem with high-temperature reactivation of the polymerase in the hot start PCR method?

High-temperature reactivation can lead to the purification of DNA templates, thereby reducing the quality of the products produced. Signup and view all the answers

What happens to the antibody in the antibody modification of the polymerase method during the denaturation step?

The antibody releases the enzyme and is denatured. Signup and view all the answers

What is an advantage of using antibody-modified polymerase compared to chemically modified polymerase?

No additional time is required in the initial denaturation step. Signup and view all the answers

What is the primary purpose of sequencing in the cloning process?

To ensure that small genetic edits such as SNPs have not been incorporated into the insert during the cloning process. Signup and view all the answers

What is the principle of asymmetric PCR?

The use of an excessive amount of single-strand (target single-strand) specific primer amplifies only a single template ssDNA. Signup and view all the answers

What is the purpose of using a 1:10 volume of primers in asymmetric PCR?

To amplify only a single template ssDNA for DNA sequencing and hybridization probing. Signup and view all the answers

What is the role of the Linear-after-the-exponential process in asymmetric PCR?

To deactivate the limited primer by higher annealing temperature to stop the amplification of dsDNA. Signup and view all the answers

What parameters are taken into consideration to optimize asymmetric PCR?

The annealing temperature, the concentration of the primers, the concentration of template, and the number of PCR cycles. Signup and view all the answers

What is the problem that can arise when using an excessive amount of a single primer in asymmetric PCR?

The creation of various problems due to primer concentration. Signup and view all the answers

What is the primary concern when it comes to the heating step in hot start PCR?

The primary concern is that the template DNA can be damaged or break down seriously due to the higher temperature for a longer time. Signup and view all the answers

How did the initial hot start PCR method limit the reaction components?

The initial hot start PCR method limited the concentration of Mg2+, dNTP, or enzyme. Signup and view all the answers

What is the purpose of using a wax bead barrier layer in hot start PCR?

The wax bead barrier layer separates the reaction components and melts when the mixture is heated during the initial denaturation step, allowing the hot start to occur. Signup and view all the answers

What is the advantage of using chemically or antibody-engineered polymerases in hot start PCR?

These polymerases are activated only when they reach a certain temperature, allowing for a more controlled hot start. Signup and view all the answers

What is the main drawback of the method that involves removing essential reaction components in hot start PCR?

This method requires additional processing steps, which can be inconvenient when performing multiple reactions and increases the risk of contamination. Signup and view all the answers

What is the purpose of the hot start in PCR, specifically in preventing nonspecific binding?

The hot start prevents the formation of new DNA from aggregating at the initial stage of the reaction, preventing nonspecific binding between primers and nonspecific DNA targets. Signup and view all the answers

What is the general requirement for most restriction enzymes to cut efficiently?

3 to 5 flanking nucleotides Signup and view all the answers

What is a useful rule of thumb when working with restriction enzymes in PCR cloning?

Add an additional restriction enzyme site outside the chosen restriction enzyme sites Signup and view all the answers

What are the general steps involved in restriction enzyme cloning after PCR amplification?

Amplification, restriction enzyme digestion, band purification, ligation, transformation, selection, and remaining steps of the cloning cycle Signup and view all the answers

What is the main purpose of site-directed mutagenesis by PCR?

To introduce specific nucleotide substitutions or deletions in a tailored manner Signup and view all the answers

What are some of the applications of site-directed mutagenesis by PCR?

Conventional cloning, mapping of regulatory elements, functional analysis of proteins, and SNP analysis Signup and view all the answers

What is an example of how site-directed mutagenesis by PCR can be used in functional analysis of proteins?

Performing alanine scanning mutagenesis or targeted substitution of key residues Signup and view all the answers

What is the primary application of site-directed mutagenesis in the context of CRISPR technology?

To introduce tailored mutations to endogenous DNA through homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. Signup and view all the answers

Why is it necessary to mutate the PAM sequence in a plasmid used for site-directed genome editing?

To render the resulting construct resistant to Cas9 induced cleavage. Signup and view all the answers

What is the role of the methylation-dependent endonuclease in the site-directed mutagenesis process?

To remove the parent template. Signup and view all the answers

What is the final step in verifying the success of site-directed mutagenesis?

Sequencing the positive clones to confirm the desired modification and the absence of additional modifications. Signup and view all the answers

What is the purpose of using a PCR protocol that amplifies the entire plasmid template in site-directed mutagenesis?

To introduce point-mutations to the plasmid using primers with the desired mutation. Signup and view all the answers

What is the advantage of using site-directed mutagenesis in genome editing?

It allows for tailored mutations to be introduced to endogenous DNA in a precise and targeted manner. Signup and view all the answers

Study Notes

Hot Start PCR Methods

Hot start PCR is used to prevent non-specific binding of primers to template DNA at room temperature
Methods of hot start PCR include:
Chelation of Magnesium Ions
- Magnesium ions are precipitated at room temperature and released at 50-95 ℃ for PCR
Chemical Modification of Polymerase
- Polymerase is reversibly inactivated by chemical modification and reactivated by preheating at 94-95 ℃ for 9-12 minutes
Antibody Modification of Polymerase
- Antibodies specific to polymerases block enzyme activity at room temperature and are denatured at high temperatures, releasing the active enzyme

Asymmetric PCR

Asymmetric PCR amplifies a single strand of template DNA for applications like DNA sequencing and hybridization probing
Uses a 10:1 ratio of target-specific primer to amplify a single-stranded template
Optimization parameters include annealing temperature, primer concentration, template concentration, and number of PCR cycles

Restriction Enzyme Cloning

PCR products are digested with restriction enzymes to create compatible ends for ligation
Typical steps include amplification, restriction enzyme digestion, band purification, ligation, and transformation

Site-Directed Mutagenesis

A technique that introduces specific nucleotide substitutions or deletions in a tailored manner
Applications include introducing or removing restriction sites, mapping regulatory elements, and functional analysis of proteins
Mutations are introduced using PCR, and the parent template is removed using a methylation-dependent endonuclease

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Description

Learn about the importance of the heating step in Hot Start PCR and how to perform it properly to avoid damaging the template DNA. Discover the different methods of limiting reaction components and using wax bead barriers to achieve hot start PCR.

Hot Start PCR: Principles and Techniques