5'RACE Cloning of The Tobacco Gene SGT1

Inquiry

With the continuous development of biotechnology, many methods and means of cloning new genes have been developed, such as map-based cloning, transposon tagging, differential display of mRNA, genome subtraction, and cDNA library screening. However, most of these methods have long experimental cycles, cumbersome technical steps, and heavy workloads. Rapid Amplification of cDNA Ends (RACE) is an effective method based on PCR to rapidly amplify the 5' and 3' ends of cDNA from low-abundance transcripts. This technology is simple, fast, and cheap, and is widely used to clone the full length of unknown genes. SGT1 is an essential component of disease resistance signaling pathways mediated by multiple plant disease resistance genes. Mutation or silencing of SGT1 results in the loss of R gene-mediated disease resistance in a variety of plants.

Principle

RACE is mainly used when the partial sequence of a gene is known. When the sequence of the middle part is known, use RNA as the template and GSP1 (gene specific primer) or oligo-d (T) as the primer to obtain cDNA through reverse transcription, and then obtain the nucleic acid sequences at both ends of the mRNA. When the sequence of one end of the gene is known, onesided PCR or anchored PCR is used. In general, the use of PCR technology to amplify the target gene from a mixture with complex components and relatively few molecules of the target gene requires two primers that can specifically bind to both ends of the amplified sequence. However, to correctly amplify an unknown sequence of a certain gene, the available data and information are strictly limited. The 3'RACE and 5'RACE methods provide effective methods to solve this problem.

5'RACE or anchored PCR technology is an effective method to isolate and determine the 5' end of a certain gene from low-copy gene material. The primer used in the synthesis of the first strand of cDNA is the antisense nucleotide sequence GSP1 of the target gene to obtain the cDNA of the target gene mRNA or the gene family mRNAs, and further obtain the complete 5' end of the target gene. After cDNA synthesis, the mRNA is degraded and the first strand of cDNA is purified from a reaction containing dNTPs, GSP1, and proteins. Then use terminal deoxynucleotidyl transferase - TDT to add the polynucleotide base "C" to the 3' end of the cDNA. Then use primer GSP2 and an anchor primer (contains two sequences: one is the anchor primer, and the other is a sequence that perfectly matches the polynucleotide base "C") to perform PCR, so that GPS2 and the unknown sequence at the 5' end of the mRNA can be amplified.

Procedures

1. Synthesis of 5'RACE-cDNA

Take 1.5 mL tube 1 (experimental group) and place it on ice, add each component according to Table 1, mix thoroughly, centrifuge briefly, and then aliquot 1/2 into tube 2 (negative control). Add 0.5 µL of reverse transcriptase to tube 1 only and mix well, centrifuge briefly, reverse transcribe at 42 °C for 30 min, and then heat bath at 70 °C for 10 min to terminate the reaction. Place on ice for 15 min, add 1 µL RNase H to each tube, and place in a water bath at 37 °C for 20 min to degrade RNA. Add 5 µL of glass milk, mix well, place at room temperature for 5 min, and centrifuge at 8000 r/min for 30 s to precipitate DNA. Discard the supernatant, add 20 µL H₂O to slowly suspend the glass milk, and leave it at room temperature for 5 min. After centrifugation at 8000 r/min for 30 s, aspirate the supernatant on ice, which is cDNA, and store it at -20 °C for later use.

Table 1. Total Reaction System 1 for The Synthesis of 5'RACE-cDNA

Component	Total RNA	Primer 5'RACE AP	10X Buffer	MgCl₂	dNTPs	DTT	H₂O
Amount	2-10 µg	2 µL	4 µL	4 µL	4 µL	4 µL	Make up to 38 µL

2. cDNA tailing

This step only adds the tail to the above tube 1 (experimental group). Add each component according to the tailing system in Table 2, mix well and centrifuge at 400 r/min for 10 min. React at 94 °C for 2~3 min, immediately place on ice for 2~3 min, add TDT and mix gently, centrifuge at 400 r/min for 10 min, react at 37 °C for 10 min, then increase to 65 °C for 10 min. The obtained product was diluted 50 times and used as template for subsequent PCR. Also dilute tube 2 (negative control group) 50 times and set aside.

Table 2. Tailing System of cDNA

Component	H₂O	5X Tailing Buffer	2 mmol/L dCTP	Recovered cDNA
Amount	1.5 µL	5 µL	2.5 µL	15 µL

3. 5'RACE-PCR1

Take two tubes and label them tube 1 and tube 2. Tube 1 uses the diluted cDNA of the experimental group synthesized above, and tube 2 uses the diluted cDNA of the control group synthesized above (negative control).

On ice, add ingredients to tube 1 and tube 2 according to the reaction system in Table 3, finally add enzyme, mix well, and centrifuge briefly. Set the PCR machine according to the reaction program in Table 4. After completing the amplification, dilute the product 50 times for later use.

Table 3. Reaction System of 5'RACE-PCR1

Component	10X Buffer	10X DMSO	dNTP	Pfu	5'RACE GSP2	AAP	cDNA	H₂O
Volume	5 µL	5 µL	2.5 µL	1 µL	1 µL	1 µL	1 µL	33.5 µL

Table 4. Reaction Program of 5'RACE-PCR1

Program of PCR1	First 5 Cycles			The Last 30 Cycles
Temperature	94 °C	48 °C	72 °C	94 °C	55 °C	72 °C
Time	1 min	40 s	1 min	1 min	40 s	1 min

4. 5'RACE-PCR2

Take two tubes and label them as tube 3 and tube 4. Tube 3 uses the diluted product of the above-mentioned PCR1 tube 1, and tube 4 uses the diluted product of the above-mentioned PCR1 tube 2 (negative control).

On ice, add each component to tube 3 and tube 4 according to the reaction system in Table 5, finally add enzyme, mix well, and centrifuge briefly. Set the PCR machine according to the reaction program in Table 6 to complete amplification.

Table 5. Reaction System of 5'RACE-PCR2

Component	10X Buffer	10X DMSO	dNTP	Pfu	5'GSP3	AP	The Diluted Product	H₂O
Volume	5 µL	5 µL	2.5 µL	1 µL	1 µL	1 µL	5 µL	28.5 µL

Table 6. Reaction Program of 5'RACE-PCR2

Program of PCR2 (35 Cycles)	94 °C	55 °C	72 °C
Time	1 min	40 s	1 min

5. Result verification

After electrophoresis of the PCR results, several bands will be obtained. Each band will be recovered and then recombined. Transform E. coli, extract and sequence the plasmid, and perform sequence analysis to obtain the correct sequence. The negative control PCR product electrophoresed without any bands.

Combined with the 3' end sequence obtained in the previous experiment, the 5' end and 3' end primers were designed respectively, and the complete SGT1 gene could be obtained by PCR amplification from the reverse transcribed cDNA.

Note:

The quality of RNA used for reverse transcription must be guaranteed. For mRNA larger than 4kb, reverse transcription needs to be performed at 42 °C for 50 min.
Reagents and supplies used for reverse transcription are RNase-free.
The activity of various enzymes used should be high.
The recovery of cDNA using glass milk should be very gentle.
Each step of cDNA synthesis and PCR must have a negative control.

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