Fidelity Assess of DNA polymerase

The Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments in vitro, widely applied in the field of life science research. However, DNA polymerase can introduce errors during the DNA synthesis process, which can lead to the amplification of incorrect sequences and affect the accuracy of results. Maintaining the accuracy of DNA sequences during PCR amplification is particularly important in applications such as cloning, site-directed mutagenesis, template preparation for sequencing, and studies of genetic variability.

There are several methods available to assess the fidelity of DNA polymerase, including Constant Denaturant Capillary Electrophoresis (CDCE), Denaturing Gradient Gel Electrophoresis (DGGE), PCR/RFLP, blue-white screening, Sanger sequencing, and Next-Generation Sequencing (NGS). However, the most common methods for assessing fidelity are blue-white screening, Sanger sequencing, and NGS. The principles and steps involved in these methods are as follows:

Blue-White Screening for fidelity determination:

This method utilizes complementation between the α-peptide (N-terminus of the enzyme) encoded by the lacZ gene and the β-galactosidase (C-terminus of the enzyme) expressed by a defective strain, leading to the formation of an active β-galactosidase that can hydrolyze X-gal and produce a blue color.

Steps:

1.     Amplify the LacZα gene using the DNA polymerase to be tested.

2.     Connect the obtained LacZα gene to a linear vector to form a recombinant plasmid.

3.     Transform the recombinant plasmid into host bacteria and plate them for cultivation.

4.     Count the number of blue and white colonies to calculate the mismatch rate.

Formula: (1 - mismatch rate) number of bases x cycle number = number of blue colonies / total colonies

Steps for blue-white screening for fidelity determination (sourced from Internet)

The commonly encountered β-galactosidase deficient strains of bacteria include DH5α, TOP10, and others. The vectors suitable for blue-white screening include the pUC series (such as pUC18, pUC19) and the M13mp series.

Sanger Sequencing for fidelity determination:

This method involves sequencing DNA from individual colonies to determine the number of mismatched nucleotides as a measure of DNA polymerase fidelity.

Steps:

1.     Amplify the DNA template using the DNA polymerase to be tested through PCR.

2.     Clone the PCR product into a vector and transform it into host bacteria for colony formation.

3.     Select individual colonies for high-throughput sequencing.

4.     Count the number of mismatched nucleotides to calculate the mismatch rate.

Formula: mismatch rate = number of mismatched nucleotides / total number of nucleotides polymerized

Steps for Sanger Sequencing for fidelity determination (sourced from Internet)

NGS Sequencing for fidelity determination:

This method involves sequencing the PCR products obtained using the DNA polymerase to be tested on an NGS platform. It relies on counting the number of mismatched nucleotides as a measure of DNA polymerase fidelity.

Steps:

1.     Design PCR primers based on the template.

2.     Perform PCR amplification using the DNA polymerase to be tested.

3.     Fragment and sequence the PCR products using a library preparation kit and NGS platform.

4.     Select and analyze the same mutation in both forward and reverse directions to calculate the mismatch rate.

Formula: mismatch rate = mutations introduced by DNA polymerase amplification / effective DNA sequencing volume

Steps for NGS Sequencing for fidelity determination (王金，2017)

Advantages and disadvantages of the three methods:

Reference:

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