3'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.

3'RACE uses poly(A) in mRNA as a primer design location in PCR amplification. In this method, reverse transcriptase and modified oligo-d(T) are used to add a specific sequence to the 5' end of oligo-d(T) to reverse-transcribe mRNA into cDNA. Then use primers that can specifically pair with the known sequence of the target gene and the specific sequence at the 5' end to amplify the specific cDNA, and you can obtain the unknown 3'-mRNA sequence of the gene between the known sequence and poly(A).

Procedures

1. Synthesis and purification of 3'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 for The Synthesis of 3'RACE-cDNA

Component	Total RNA	Primer 3'RACE AP	10X Buffer	MgCl₂	dNTPs	DTT	H₂O
Amount	1-5 µg	1 µL	2.5 µL	2.5 µL	4 µL	2.5 µL	Make up to 24 µL

2. 3'RACE-PCR1

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

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

Table 2. Reaction System of 3'RACE-PCR1

Component	10X Buffer	10X DMSO	dNTP	Pfu	3'GSP1	AUAP	cDNA	H₂O
Volume	5 µL	5 µL	2.5 µL	1 µL	1 µL	1 µL	1 µL	33.5 µL

Table 3. Reaction Program of 3'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

3. 3'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 4, finally add enzyme, mix well, and centrifuge briefly. Set the PCR machine according to the reaction program in Table 5. After completing the amplification, dilute the product 50 times for later use.

Table 4. Reaction System of 3'RACE-PCR2

Component	10X Buffer	10X DMSO	dNTP	Pfu	3'GSP2	AUAP	The Diluted Product	H₂O
Volume	5 µL	5 µL	2.5 µL	1 µL	1 µL	1 µL	1 µL	33.5 µL

Table 5. Reaction Program of 3'RACE-PCR2

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

4. 3'RACE-PCR3

Take two tubes and label them as tube 5 and tube 6. Tube 5 uses the diluted product of the above-mentioned PCR2 tube 3, and tube 6 uses the diluted product of the above-mentioned PCR2 tube 4 (negative control).

On ice, add ingredients to tube 5 and tube 6 according to the reaction system in Table 6, finally add enzyme, mix well, and centrifuge briefly. The PCR3 reaction procedure is the same as the PCR2 reaction procedure.

Table 6. Reaction System of 3'RACE-PCR3

Component	10X Buffer	10X DMSO	dNTP	Pfu	3'GSP3	AUAP	The Diluted Product	H₂O
Volume	5 µL	5 µL	2.5 µL	1 µL	1 µL	1 µL	1 µL	33.5 µL

5. Result verification

The result of 3'RACE-PCR3 is to obtain a DNA fragment of 300 bp. The negative control PCR product showed no bands during electrophoresis. The obtained fragment was cloned, transformed into E. coli, and the plasmid was extracted and sequenced to obtain the correct sequence fragment.

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.

Related Services

Our products/services are For Research Use Only. Not For Clinical Use!

Online Inquiry