Analyzing substrates and products of ligation reactions by PCR

Summary

Synthetic DNA libraries generated by PCR can be digested enzymatically and ligated into a linearized expression vector system. In the scheme presented here, the 5' (Not I) and 3' (Cla I) ends of the library are different cleavage sites and the same cleavage sites are used for cloning into the vector. The efficiency of the linearization and ligation reactions can be analyzed by PCR. This experiment is from PCR Lab Guide (2nd edition) by Seed Kang and Qu Lijia.

Operation method

Analyzing substrates and products of ligation reactions by PCR

Materials and Instruments

ddH2O Vent buffer Vent DNA polymerase dNTP primers Plasmid DNA
Reagents and equipment for agarose gel electrophoresis Thermocycler

Move

I. Materials

1. Buffers, solutions and reagents

_ddH2O

2. enzymes and enzyme buffers

10X Vent buffer

Vent DNA Polymerase

3. Nucleic acids and oligonucleotides

dNTP, 20 mmol/L each

primers that can anneal to vector sequences flanking the cloning site of the synthesized DNA library

Plasmid DNA (see step 1 below)

4. Specialized equipment

Reagents and equipment for agarose gel electrophoresis, including ethidium bromide

Thermocycler

II. Methods

1.Mix the following components.

10~50ng of plasmid DNA

50pmol of each primer

5ul of 10X Vent buffer

dATP, dCTP, dTTP and dGTP, 0.5ul each

1U of Vent DNA Polymerase

Vent DNA Polymerase, 0.5ul of each of _dATP, dCTP, dTTP and dGTP.

Each of the following plasmid vectors should be reacted separately:

Undigested vector

Digested vector, but not ligated

Vector digested and ligated without library DNA

Vector digested and ligated with library DNA

2. Preheat the mixture for 20s at 94°C in a thermal cycler.

3. Perform 30 rounds of PCR cycles according to the following parameters.

94°C10s

55°C10s (or appropriate annealing temperature with primers)

72°C15s

Extend the extension time of the last cycle to 30s

4. Cool to 4°C

5. Take 25ul of each reaction product, run 4% agarose gel electrophoresis and analyze with ethidium bromide staining (Sam-brook and Russell 2001).

Analysis

In the example shown in Figure 26-3, PCR analysis of the linearized vector prior to ligation showed that the vector was fully linearized because no PCR product corresponding to the starting vector was obtained (Figure 26-3, lane 4). In the absence of the library insert, PCR of the ligated vector showed that a certain amount of self-association had occurred (Figure 26-3, lane 2) and that a small portion of the vector was singly cleaved. However, when the vector was ligated to the library insert, the majority of the DNA product corresponded to the correctly ligated library DNA and there was no PCR product corresponding to the starting vector (Figure 26-3, lane 3). When the vector is ligated to the insert, the PCR product corresponding to the starting vector is not detected, but it is detected only when the vector is ligated (compare Figure 26-3, lanes 2 and 3), which can be explained by the fact that the library DNA is ligated to the singly cleaved vector. This by-product does not form a closed circular plasmid and cannot be cloned, nor does it produce a PCR product.



In this case, PCR is an important analytical tool that can reliably validate restriction and ligation reactions. As a result, the researcher can use the correct DNA for the subsequent steps of library construction and is more likely to end up with a high-quality library. The final library can be obtained by transforming and amplifying the bacteria and then purifying the plasmid DNA using standard procedures (Sam brook and Russell 2001).

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