Rapid amplification of cDNA 5' ends (5'-RACE)
Rapid amplification of cDNA 5' ends (5'-RACE)
There is no greater failure in molecular cloning than the isolation of a cDNA clone that lacks the characteristic structure of the sequence at the 5' end of the corresponding mRNA. The first strand of the synthesized cDNA is often incomplete at the 5' end due to the failure of the reverse transcriptase to extend completely along the full-length mRNA template, as is often the case with some of the clones present in cDNA gene libraries. This experiment is from "Molecular Cloning Laboratory Guide, Third Edition", translated by Huang Peitang et al.
Operation method
Rapid amplification of cDNA 5' ends (5'-RACE)
Principle
There is no greater failure in molecular cloning than the isolation of a cDNA clone that lacks the characteristic structure of the sequence at the 5' end of the corresponding mRNA. Some of the clones usually present in cDNA gene libraries often synthesize cDNAs with incomplete 5' ends on the first strand because the reverse transcriptase enzyme fails to extend completely along the full-length mRNA template.
Materials and Instruments
Reverse transcriptase Terminal deoxynucleotidyl transferase Terminal transferase buffer Heat-stable DNA polymerase Move I. Materials For more product details, please visit Aladdin Scientific website.
Amplification buffer Chloroform dATP dNTP storage solution Placenta RNase inhibitor TE Reverse transcriptase buffer
Agarose or polyacrylamide gel Preparative centrifuge SorvallSS-34 head
1. Buffers and solutions
10X Amplification Buffer
Chloroform
dATP ( 1 mmol/L, disodium salt)
4 dNTP storage solutions (20 mmol/L, pH 8.0)
Placental RNase inhibitor (20 units/μl)
TE (pH 7.6)
2. Enzyme and buffer
5X reverse transcriptase buffer
Reverse transcriptase (RNA-dependent DNA polymerase)
Terminal deoxynucleotidyl transferase (terminal transferase)
5X terminal transferase buffer
Heat Stable DNA Polymerase
3. gels
Agarose gel or polyacrylamide gel
4. nucleotides and oligonucleotides
Junction primer (10 μmol/L, 5'GACTCGAGTCGACATCG3') dissolved in water (10 pmol/μl)
(Splice primers can be used in combination with gene-specific forward primers to amplify specific target cDNAs.) After the first step of PCR amplification, the PCR target product may represent as little as 1% of the total DNA product or as much as 100% of the total DNA product. If necessary, the yield of the target product can be improved by using the product of the first round of PCR as a template for a second round of nested PCR, which consists of a junction primer and another gene-specific forward primer. After the second round of nested PCR amplification, almost all amplified products showed bands consistent with the sequence of the 5' region of the target mRNA under EB staining.
Primers for RT-PCR are synthesized using an automated DNA synthesizer, and primers for standard RT-PCR generally do not require further purification. However, if oligonucleotide primers are purified by column chromatography with commercial resins (e.g., NENSORB from NENLife-Science) or by denaturing polyacrylamide gel electrophoresis, the purified primers are often more efficient when used to amplify low-abundance mRNAs).
(dT )17-junction primer (10 μmol/L, 5'GACTCGAGTCGACATCGA(T )173 ') was dissolved in water (10 pmol/μl).
Gene-specific antisense oligonucleotide primer (10 μmol/L) dissolved in water (10 pmol/μl).
(Gene-specific antisense oligonucleotide primers should be complementary to the known sequence of the target mRNA, with a length of 20~30 bp, containing approximately equal numbers of the four bases, a balanced distribution of G and C bases, and a low tendency for the primers to spontaneously form stable secondary structures. Gene-specific primer 1 is used in step 2 to guide the synthesis of gene-specific first-strand cDNA by reverse transcriptase. Gene-specific primer 2 is complementary to the 5' sequence of the target mRNA and is used in the amplification phase of the 5'-RACE reaction. Gene-specific primer 2 is also always inserted into the appropriate restriction endonuclease cleavage site, which facilitates further cloning of the amplified cDNA).
Random hexanucleotides are dissolved in TE ( 1 mg/ml, pH 8.0)
Total RNA (100 μg/ml) or poly (A )+ RNA (10 μg/ml) dissolved in water
(Total RNA is generally extracted from cells using chaotropic agents, and the choice of total RNA as a template is generally applicable to the cloning of target genes by RT-PCR from moderate to high abundance mRNA. For samples with low abundance of target mRNA, poly (A) + RNA is preferred as a template for RT-PCR. RNA utilized as a template for RT-PCR can also be prepared from a small number of cultured cells as described below).
5. Centrifuges and Rotors
Preparative Centrifuge
SorvallSS - 34 Heads
6. Specialized equipment
Shielded tips for automated micropipettes
Concentrators (Centricon-100, Amicon products)
Microcentrifuge tubes (0.5 ml, special centrifuge tubes for thin-walled amplification reactions) or microtitre plates
Positive displacement pipettes
PCR instrument
Rotary Vacuum Freeze Dryer (optional)
Water bath preset at 75°C and 80°C
II. Methods
Reverse transcription
At the beginning of the experiment, the first step is to determine whether the 5'-RACE is successful. If the reverse transcription step is effective, the chances of isolating a clone containing the 5' end sequence of the target mRNA are high. On the other hand, if the reverse transcription step is ineffective, then the subsequent steps of this protocol are not compensated. Therefore, it is worthwhile to spend time optimizing the reaction conditions for reverse transcription, such as the optimal primer-to-template ratio, and varying the Mg2+ concentration of the reaction system to find the optimal Mg2+ concentration.
1. Transfer 1 pg to 100 ng of poly (A) + RNA or 1 μg of total RNA to a new centrifuge tube. Adjust the volume with water to denature the RNA sample at 75°C for 5 min, and cool the tube in ice.
2. Add the following reagents sequentially to the centrifuge tube containing the denatured RNA sample:
5X Reverse Transcriptase Buffer 4 μl
20 mmol/L 4 dNTP mix (pH 8.0) 1 μl
10 μmol/L gene-specific antisense primer 1 4 μl
Approx. 20 units/μl Placenta RNase Inhibitor 1 μl
Make up to 20 μl with water
Reaction tubes were incubated for 60 min. 3 negative control tubes were set up. In one control tube, add all reagents necessary for the synthesis of the first-strand cDNA reaction, but no template RNA; in the second control tube, add all reagents except reverse transcriptase; and in the third control tube, add all reagents except primers. These control tubes perform all subsequent steps. The setup of these control tubes ensures that the cDNA product is not produced by contamination of genomic DNA and oligonucleotide fragments or by self-direction of the RNA template molecules.
Purification of the reverse transcription product
Upon completion of the reverse transcription reaction, excess dNTP and primers must be removed from the reaction solution. In addition, the presence of dNTP in the reaction solution may, on the one hand, be caused by the tailing of the 3' end of the cDNA by the terminal transferase, and on the other hand, it may jeopardize the ability of the first-strand cDNA tail sequence to serve as a primer binding site. The presence of excess primers can also be detrimental, as the 3' end of the primer competes with the cDNA tailing reaction.
3. Removal of excess oligonucleotide or random hexanucleotide primers can be accomplished by diluting the final volume of the reaction solution to 2 ml with water, and then concentrating the reaction solution using a Centicon-100 microconcentrator (Frohman 1993; for a description of this procedure, please see protocol 3). Centrifuge at 500-1100 g (2000-3000 r/min, Sorvall SS34 head) for 20 min at temperatures between 4°C and 25°C. Repeat the dilution with water and centrifugation steps. Transfer the trapped liquid to a new 0.5 ml centrifuge tube and concentrate the volume to 10 μl using a rotary vacuum extractor.
4. Add 10 volumes of the following reagents to the reverse transcribed cDNA sample:
5X terminal transferase buffer 4 μl
1 mmol/L dATP 4 μl
Terminal transferase 10~25 units
The reaction tubes were incubated in a 37℃ water bath for 15 min.
5. Inactivate the terminal transferase by placing at 80°C for 3 min. Dilute the cDNA sample with dA tail with TE pH 7.6 to a final volume of 1 ml.
Amplification
6. Create a PCR series by adding the components to the wells of a 0.5 ml sterilized centrifuge tube, amplification tube or sterilized microtitre plate in the following order:
Diluted cDNA 0-20 μl
10X amplification buffer 5 μl
20 mmol/L 4 dNTP mix 5 μl
10 μmol/L (dT )17-junction primer (16 pmol) 1.6 μl
10 μmol/L junction primer (32 pmol) 3.2 μl
10 μmol/L gene-specific primer 2 ( 32 pmol) 3.2 μl
1~2 units of heat stabilized DNA polymerase 1 μl
H2O make up to 50 μl
If necessary, a series of amplification test tubes can be set up to screen cDNA tailing templates to produce the largest number of amplified 5' ends.
7. If the PCR instrument is not equipped with a heated lid, a drop of mineral oil (approx. 50 μl) should be added 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 drop of paraffin oil to the top of the reaction mixture. Place a centrifuge tube or microtitre plate on the PCR instrument.
8. Perform PCR amplification as follows. Typical procedures are denaturation, denaturation and polymerization (extension reaction); the corresponding cycling conditions and temperatures are listed below: 
9. 5-10 μl of each amplified sample is withdrawn, and the results are analyzed by agar 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.
10. If mineral oil is used to cover the top layer of sample liquid in the microcentrifuge tube (step 7), it can be removed by 150 μl of chloroform extraction at the end of the reaction.
11. Separate the residual heat-stable DNA polymerase and dNTP from the amplified DNA product. 