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| This protocol describes the 5'-end amplification of mRNA by nested PCR. 5' RACE allows for the synthesis of unknown sequences at the 5' end of a mRNA transcript. First strand cDNA is synthesized from total or poly(A+) RNA using a gene-specific primer. After the first-strand cDNA is purified, a tail of polyA or polyG is added to the 3' end. This "tailed" cDNA is then amplified in a PCR reaction using a second, nested gene-specific primer and an oligo (dT) or (dC) adaptor. |
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A. Synthesis of the First Strand of cDNA Using a Gene-specific Primer
1. Dissolve 1 to 5 μg of poly (A)+ RNA or 10-20 μg of total RNA in 10 μl of DEPC-treated ddH2O (see Hint #1).
2. Heat at 65°C for 5 min.
3. Chill on ice.
4. Add:
2 μl of 10X PCR Buffer
2 μl of 10 mM dNTPs
2 μl of DTT
0.5 μl of RNasin
3 μl of Gene-specific Primer 1
1 μl of AMV Reverse Transcriptase (see Hint #2)
5. Incubate the reaction at 42°C for 1 to 2 hr.
6. Heat the reverse transcriptase mixture at 95°C for 5 min.
7. Dilute the reaction with 2 ml of ddH2O and transfer to a Centricon-100 (100,000 Nominal MW cutoff) Centrifugal Filter Unit.
8. Centrifuge at 1,500 X g (for example at 3,500 rpm in a SS34 rotor for 45 min to remove excess Primer 1 (see Hint #3).
9. Repeat Steps #7 and #8 and collect retained liquid.
B. Tailing the 3' End of the Target cDNA
1. Heat 15 μl aliquots of the first-strand cDNA prepared in Section A at 65°C for 5 min.
2. Chill on ice.
3. Add:
2 μl of 10X Tailing Buffer
2 μl of 10 mM dGTP (or dATP)
1 μl of Terminal Transferase
4. Incubate the reaction mixture at 37°C for 10 to 15 min.
5. Heat at 65°C for 10 min.
6. Add 200 μl of TE.
7. Add an equal volume of Phenol/Chloroform to extract. Vortex to mix.
8. Centrifuge sample in microcentrifuge for 3 min at room temperature to separate the phases.
9. Collect the upper, aqueous phase.
10. Dilute the mixture with 2 ml water and transfer to a Centricon-100 Centrifugal Filter Unit.
11. Centrifuge at 1,500 X g for 45 min.
C. Amplification of the Tailed cDNA by PCR
1. Perform the PCR as above, using:
10 μl of the tailed cDNA
4 μl of Gene-specific Primer 2
4 μl of (dC)15-adaptor, if the template was tailed by dGTP,
- OR 4 μl of (dT)17-adaptor, if the template was tailed by dATP
10 μl of 2 mM dNTPs
Add ddH2O to 100 μl
Then add 0.5 μl of Taq polymerase
2. Run a PCR program with 40 cycles in a thermal cycler (see Hint #4)
3. Combine10 μl aliquots of PCR products with 5 μl of Sucrose Loading Dye.
4. Load the samples on an agarose gel for analysis of the reaction products (see Hint #5 and see Protocol on Running DNA on Agarose Gels).
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| Sucrose Loading Dye (6X) |
| 0.25% Bromophenol Blue
40% (w/v) Sucrose
0.25% Xylene Cyanol
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| Gene-specific Primer 2 |
| 50 ng/μl anti-sense primer to gene of interest located upstream of Gene-specific Primer 1
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| Gene-specific Primer 1 |
| 50 ng/μl anti-sense primer to gene of interest
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| (dT)17-adaptor |
| 50 ng/μl (dT)17-adaptor
5'-GAC TCG AGT CGA CAT CGA TTT TTT TTT TTT TTT TT-3'
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| (dC)15-adaptor |
| 5'-AAA AGA TCT GTC GAC CCC CCC CCC CCC CC-3'
50 ng/μl (dC)15-adaptor
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| 10 mM DTT |
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| 10 mM dNTPs |
| 10 mM dATP
10 mM dTTP
10 mM dGTP
10 mM dCTP
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| Phenol/Chloroform |
| Store at 4°C in a dark glass bottle
25:24:1 Phenol:Chloroform:Isoamyl Alcohol (CAUTION! see Hint #6)
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| 10X PCR Buffer |
| 15 mM MgCl2
500 mM KCl
100 mM Tris-Cl, pH 8.3
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| 10X Tailing Buffer |
| 2 mM DTT
250 mM Tris-Cl, pH 7.6
10 mM Cobalt Chloride
1 M Potassium Cacodylate
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| 10 mM dGTP or 10 mM dATP |
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| 2 mM dNTPs |
| 2 mM dGTP
2 mM dCTP
2 mM dATP
2 mM dTTP
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Phenol
Tris
Potassium Chloride
Cobalt Chloride
DTT
Xylene Cyanol
Bromophenol Blue
DNA Polymerase, Taq
Two gene-specific primers
(dT)17-adaptor of (dC)15- adaptor
Potassium Cacodylate
Oligonucleotide
Terminal Transferase
AMV Reverse Transcriptase
Sucrose
dCTP
dTTP
dGTP
dATP
Magnesium Chloride
RNasin
Isoamyl Alcohol
Chloroform
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1. To minimize the degradation of RNA by RNases, wear gloves when handling samples and reagents and change gloves regularly while working. Treat water and solutions with DEPC* (diethyl pyrocarbonate) to inactive RNases and use solutions prepared with RNase-free water and equipment. Use sterile plasticware or glassware that have been baked at 150°C in dry heat for at least 2 hours. Autoclaving of solutions and vessels is insufficient to remove all RNase activity. Do not use spatulas to measure out chemicals used for RNAse-free solutions. Reserve RNase-free solutions, reagents, and consumables, such as pipette tips, only for RNA work. Some researchers find that following all of the above measures is not necessary for good quality RNA preps. You must determine for your own work how conservative to be in eliminating sources of contaminating RNases. To treat water with DEPC
Add DEPC to dd water to a final concentration of 0.1% (v/v). Shake up or stir solution. Allow solution to sit for at least 12 hours. Autoclave for 15 minutes to inactivate the DEPC. CAUTION! DEPC is a carcinogen; handle appropriately.
2. AMV reverse transcriptase is used because it is stable at higher temperatures than other reverse transcriptases. This allows for the reaction to proceed at 55°C, if so desired, to eliminate potentially inhibitory RNA secondary structures.
3. There is no need to purify the cDNA because the RNAse H activity of the AMV reverse transcriptase degrades the RNA template.
4. Cycle conditions will depend on your primer, the size of the PCR product, etc. Optimize the PCR for your particular application.
5. Southern blot analysis can also be performed using an internal gene-specific probe.
6. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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1. Frohman, MA, Dush, MK, Martin, GR. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 1988; 85:8998-9002.
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