Long-range PCR

Summary

Under standard PCR reaction conditions, PCR amplification of DNA fragments is generally 1-2 kb in length, which is sufficient for many routine DNA molecular manipulation techniques (e.g., DNA sequence analysis and gene mutation, etc.), but for amplification of certain large and complete mammalian genomes, such PCR amplification is far from meeting the experimental requirements. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.

Operation method

Long-range PCR

Principle

Under standard PCR reaction conditions, PCR amplifies DNA fragments of 1-2 kb in length, which is sufficient for many routine DNA molecular manipulation techniques (e.g., DNA sequence analysis and gene mutation, etc.), but it is far from sufficient for amplification of some large complete mammalian genomes, or even medium-sized genomes, and for an average-length full-length dDNA. However, for amplification of some large complete mammalian genomes, even medium-sized genomes, and for an average-length full-length dDNA, this PCR amplification capability is still far from being adequate.

Materials and Instruments

DNA template Forward primer
Chloroform dNTP storage solution Long-range PCR buffer Heat-stabilized DNA polymerase mixture
Agarose gel Shielded tip Tubes Positive displacement pipettes PCR instruments

Move

I. Materials

1. Buffers and solutions

Chloroform

4 dNTP storage solutions (20 mmol/L, pH 8.0)

10X long-range PCR buffer (500 mmol/L Tris-Cl ( pH 9.0, room temperature), 160 mmol/L ammonium sulfate, 25 mmol/L MgCl₂, 1.5 mg/ml bovine serum albumin)

2. Enzyme and buffer

Heat-stabilized DNA polymerase mix

3. gels

Agarose gel

4. nucleotides and oligonucleotides

DNA templates

Forward primer (20 μmol/L) and reverse primer (20 μmol/L) were dissolved in water.

5. Special equipment

Shielded tips for automated micropipettes.

Centrifuge tubes (0.5 ml, thin-walled for amplification reactions)

Positive displacement pipettes

PCR instrument

II.

1. Using a thin-walled amplification centrifuge tube, add the following reagents in sequence and mix:

10X Long Range PCR Amplification Buffer 5 μl

20 mmol/L 4 dNTP mix 5 μl

20 mmol/L forward primer 1 μl

20 mmol/L reverse primer 1 μl

Heat-stabilized DNA polymerase mix 0.2 μl

DNA template 100 pg~2 μg

_H2O make up to 50 μl

2. Add a drop of clear mineral oil (approx. 50 μl) to the top of the reaction mixture if the PCR is not equipped with a heated lid, or add a layer of paraffin oil to the top of the reaction mixture if the PCR program is to be started hot. Place a centrifuge tube or microtitre plate on the PCR instrument. Perform PCR amplification as follows. Typical procedures are denaturation, denaturation and polymerization (extension reaction); the corresponding cycling conditions and temperatures are listed below:



3. If mineral oil is used to cover the top layer of sample liquid in the microcentrifuge tube (step 2), it can be removed by 150 μl of chloroform extraction at the end of the reaction.

4. Several samples of the aqueous phase of the amplification product are drawn and analyzed by agarose gel electrophoresis supplemented with an appropriately sized DNA marker. In many cases, the amplification product is so small that the target band cannot be detected by conventional ethidium bromide staining. In such cases, staining of the DNA sample on the gel with SYBR gold particles or transferring the DNA sample on the gel to a nylon or nitrocellulose filter membrane for Southern hybridization with the probe is used to confirm the results.

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