Question

The plasmid that you received was ~4500 bp and among other things had our

gene of interest inserted downstream of a T7-promoter. Following the PCR

reaction how big was the predominant piece of DNA that was amplified? How

does the size of this PCR product compare to the PCR product from the

experiment performed at the very beginning of the lab (

2 points

)?

i. Size of site-directed mutagenesis PCR product:

ii. Size of beginning of the semester PCR product:

Answer 1

T7 polymerase is extremely promoter-specific and transcribes only DNA downstream of a T7 promoter. The T7 polymerase also requires a double stranded DNA template and Mg²⁺ ion as cofactor for the synthesis of RNA. It has a very low error rate.This blocks access of T7 RNA polymerase to the promoter site and thus prevents leaky transcription of your gene before induction. When lactose binds to LacI it induces a conformational change in the protein structure that renders it incapable of binding to the operator DNA sequence.

Site directed mutagenesis is a highly versatile technique that can be used to introduce specific nucleotide substitutions (or deletions) in a tailored manner. The approach can be used in conventional cloning (to introduce or remove restriction sites), in mapping of regulatory elements (to mutate promoters/enhancers in reporter constructs), in functional analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key residues), and in SNP analysis (to introduce naturally occuring SNPs in a plasmid context). The technique is also highly relevant in this age of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate genome editing. Tailored mutations are commonly introduced to endogeneous DNA through homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed genome editing requires a template of high homology to the endogenous target, yet to facilitate the repair, the template should be resistant to Cas9 cleavage. If a plasmid contains the template, site-directed mutagenesis can be used to mutate the PAM sequence (an NGG sequence critical for Cas9 cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage.

In brief, point-mutations can be introduced to plasmids using primers (with the desired mutation) in a PCR protocol that amplifies the entire plasmid template. The parent template is removed using a methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked plasmid (the PCR product). Plasmids are isolated from the resulting colonies, and screened for the desired modification. Finally, the positive clones are sequenced to confirm the desired modification and the absense of additional modifications.

Answer 2

To determine the size of the predominant piece of DNA amplified by PCR, we need more information about the specific primers used in the reaction. The size of the PCR product depends on the primer sequences and the location of the primers relative to the gene of interest.

Without information about the primer sequences used in the PCR reaction for both the site-directed mutagenesis and the beginning of the semester experiment, it is not possible to calculate the exact size of the PCR products.

However, we can make some general observations:

i. Size of site-directed mutagenesis PCR product: Since the plasmid received was ~4500 bp and the gene of interest was inserted downstream of a T7-promoter, the PCR product in the site-directed mutagenesis experiment would depend on the primers used and the specific region targeted. If the primers were designed to amplify only the gene of interest downstream of the T7-promoter, the PCR product would likely be smaller than 4500 bp.

ii. Size of the beginning of the semester PCR product: Similarly, without information about the primers used at the beginning of the semester, we cannot determine the exact size of the PCR product. The size of the PCR product in the beginning of the semester experiment would also depend on the primers used and the specific target sequence.

In summary, to determine the size of the PCR products, we need the specific primer sequences used in both experiments. Without this information, we can only make general observations based on the design of the experiments and the known size of the plasmid and gene of interest.