Question

When altering a 7-base pair seed sequence in the middle of an amplicon for synthetic biology purposes, what method is commonly used?

A) PCR mutagenesis with flanking homology regions.
B) Site-directed mutagenesis with no size constraints.
C) Purchasing gBlock for efficient sequence alteration.
D) Employing CRISPR-Cas9 technology for precise modifications.

What is the primary purpose of having sufficient flanking homology regions in the PCR mutagenesis method?

A) To increase the overall length of the amplicon.
B) To facilitate primer binding and ensure specificity.
C) To eliminate the need for flanking homology.
D) To enable direct synthesis of the modified sequence.

Answer 1

The commonly used method for altering a 7-base pair seed sequence in synthetic biology is site-directed mutagenesis, due to its precision and lack of size constraints. The primary purpose of flanking homology regions in PCR mutagenesis is to facilitate primer binding and ensure specificity during the amplification process.

Step-by-step explanation:

When altering a 7-base pair seed sequence in the middle of an amplicon for synthetic biology purposes, the commonly used method is site-directed mutagenesis with no size constraints. Site-directed mutagenesis is a technique that allows for the introduction, deletion, or change of specific nucleotides within a DNA sequence. This method is particularly useful when changes are needed in a precise location and when the changes are limited to a few nucleotides. Among the common variants of this method is the QuickChange site-directed mutagenesis. It is an efficient way to perform mutagenesis without the need for restriction enzymes or ligation, thereby speeding up the process.

The primary purpose of having sufficient flanking homology regions in the PCR mutagenesis method is to facilitate primer binding and ensure specificity. Adequate homology between the primer and the target sequence ensures high efficiency in the amplification of the correct DNA segment, minimizing off-target effects and ensuring the desired alteration is achieved.