Application of mixed oligonucleotide primer-guided cDNA amplification (MOPAC)
Application of mixed oligonucleotide primer-guided cDNA amplification (MOPAC)
The best way to clone a protein for which only part of the sequence is known is to design an oligonucleotide using the known amino acid sequence and use this oligonucleotide as a probe to screen a gene library for the full-length gene or to use the oligonucleotide as a primer for PCR amplification of the target gene. Both of these methods have problems with the concatenation of the genetic codons encoded by the oligonucleotide. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Application of mixed oligonucleotide primer-guided cDNA amplification (MOPAC)
Principle
The best way to clone a protein for which only part of the sequence is known is to design the corresponding oligonucleotide using the known amino acid sequence, and use this oligonucleotide as a probe to screen the gene library for the full-length gene or to use the oligonucleotide as a primer for PCR amplification of the target gene. Both of these methods encounter the problem of concatenation of the genetic codons encoded by the oligonucleotides.
Materials and Instruments
Heat-stable DNA polymerase DNA templates Antisense oligonucleotide libraries Justified oligonucleotide primer libraries Move I. Materials For more product details, please visit Aladdin Scientific website.
Amplification buffer dNTP storage solution
Polyacrylamide gels Shielded tips Tubes Positive displacement pipettes PCR instruments
1. Buffers and solutions
10X Amplification Buffer
4 dNTP storage solutions (20 mmol/L, pH 8.0 )
2. Enzyme and buffer
Heat-stabilized DNA polymerase
3. gels
Polyacrylamide gel
4. Nucleotides and oligonucleotides
DNA template (100 μg/ml, genomic DNA or cDNA gene library) dissolved in TE (pH 8.0)
Antisense oligonucleotide library (10 μmol/L) in TE (pH 8.0)
Positive oligonucleotide primer library (10 μmol/L) in TE (pH 8.0)
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. Add the components to the wells of a 0.5 ml sterilized centrifuge tube, amplification tube or sterilized microtitre plate in the following order and mix:
0.5 μg template DNA 5 μl
10X Amplification Buffer 5 μl
20 mmol/L 4 dNTP mix (pH 8.0) 5 μl
10 μmol/L Justified Primer Library (70 pmol) 7 μl
10 μmol/L antisense primer library (70 pmol) 7 μl
1 to 2 units of heat-stabilized DNA polymerase 1 μl
H2O make up to 50 μl
Set up several control reaction tubes. In one control tube, add all of the above reagents except the template DNA. In the second tube, add all the above reagents except one primer.
2. If the PCR instrument is not equipped with a heated lid, add a drop of mineral oil (approx. 50 μl) to the top of the reaction mixture to prevent evaporation of the sample during the multiple heating and cooling cycles of the PCR reaction. If a hot-start PCR program is applied, add a layer of paraffin oil to the top of the reaction mixture. Place the centrifuge tube and microtiter plate on the PCR instrument.
3. Perform PCR amplification as follows. Typical procedures are denaturation, denaturation and polymerization (extension reaction); the corresponding cycling conditions and temperatures are listed below: 
4. 5-10 μl of each amplified sample is taken and analyzed by agarose gel electrophoresis or polyacrylamide gel electrophoresis, and a DNA marker is used to determine the size of the amplified fragments. The gel is usually stained with EB (ethidium bromide) or SYBR gold particles to observe the amount of amplification and fragment size.
If both positive and negative primers are designed for a contiguous amino acid sequence to be used in the MOPAC reaction, the size of the target amplification product will be known. In most cases, thin polyacrylamide gels (<6%) can be used to separate the nucleotide sequences in a hierarchical manner that can discriminate the size of the nucleotide sequence to allow definitive identification of the target product. One method of identification is for the amplified product to be end-labeled and its chemical sequence analyzed to determine a unique sequence that binds to both sets of oligonucleotide libraries. This unique sequence can ultimately be synthesized and used as a probe to isolate and screen for longer cDNA genes. Alternatively, the MOPAC product itself can be labeled and used as a probe in a second round of PCR. If the cleavage site is designed to be at the 5' end of the starting oligonucleotide primer, this amplified fragment can be easily cloned into the appropriate plasmid or phage vector after cleavage by the appropriate endonuclease.
5. If mineral oil is used to cover the top layer of sample liquid in the microcentrifuge tube, it can be removed by 150 μl of chloroform extraction at the end of the reaction.
Inside the PCR reaction tube, the interface between the aqueous phase containing the amplified DNA fragments and the upper layer of mineral oil forms a crescent, and the aqueous phase underneath the crescent also contains microgels. The aqueous phase can be carefully pipetted into a new centrifuge tube.
For many purposes, such as purification of amplified DNA fragments using the Cemricon Microconcentrator or cloning of amplified products, it is necessary to remove the oil phase from the upper layer of the reaction tube before proceeding with the experiment. 
