RACE System for Rapid Amplification of cDNA Ends

互联网2013-11-13

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实验原理

The 5' RACE System is a set of prequalified reagents intended for synthesis of first strand cDNA, purification of first strand products, homopolymeric tailing, and preparation of target cDNA for subsequent amplification by PCR. Control RNA, DNA, and primers are provided for monitoring system performance.

First strand cDNA is synthesized from total or poly(A) RNA using a gene-specific primer (GSP1) that the user provides and SuperScript™ II, a derivative of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) with reduced RNase H activity. After first strand cDNA synthesis, the original mRNA template is removed by treatment with the RNase Mix (mixture of RNase H, which is specific for RNA:DNA heteroduplex molecules, and RNase T1). Unincorporated dNTPs, GSP1, and proteins are separated from cDNA using a S.N.A.P. Column. A homopolymeric tail is then added to the 3'-end of the cDNA using TdT and dCTP.

Since the tailing reaction is performed in a PCR-compatible buffer, the entire contents of the reaction may be directly amplified by PCR without intermediate organic extractions, ethanol precipitations, or dilutions. PCR amplification is accomplished using Taq DNA polymerase, a nested, gene-specific primer (GSP2, designed by the user) that anneals to a site located within the cDNA molecule, and a novel deoxyinosine-containing anchor primer (patent pending) provided with the system.

Following amplification, 5' RACE products can be cloned into an appropriate vector for subsequent characterization procedures, which may include sequencing, restriction mapping, preparation of probes to detect the genomic elements associated with the cDNA of interest, or in vitro RNA synthesis. The Abridged Anchor Primer (AAP), Abridged Universal Amplification Primer (AUAP), Anchor Primer (AP) [available separately], and Universal Amplification Primer (UAP) include recognition sequences for Mlu I, Sal I, and Spe I to facilitate restriction endonuclease cloning of RACE products.

5' RACE products may also be used for procedures that do not require an intermittent cloning step such as dsDNA cycle sequencing or probe preparation. However, additional rounds of PCR using the AUAP, or UAP, in conjunction with either progressively nested GSPs or size-selected products from the initial PCR, may be required to confer an adequate level of specificity to the process to permit direct characterization of RACE products. Details of the individual steps of 5' RACE are discussed below. Review this information carefully before beginning. The RNA isolation, design of primers, and the amplification protocols are most important for optimal results.

实验步骤

1. 1X Wash Buffer for S.N.A.P. Procedure

1) Pipette 1 ml of the wash buffer concentrate into a 50-ml graduated cylinder.

2) Add 18 ml of distilled water and 21 ml of absolute ethanol. Mix thoroughly.

3) Transfer to an appropriate-sized glass bottle. Cap and store at 4°C.

2. 70% Ethanol Wash for S.N.A.P. Procedure

1) Add 35 ml of absolute ethanol and 15 ml of distilled water to a 50-ml graduated cylinder.

Table 1

Component	Amount
GSP1	2.5 pmoles (~10 to 25 ng)
^{Sample RNA}	1-5 μg
DEPC-treated water (or sterile, distilled water)	sufficient for a final volume of 15.5 μl

2) Transfer to an appropriate-sized glass bottle. Cap and store at 4°C.

3. First Strand cDNA Synthesis

1) Add the following to a 0.5-ml microcentrifuge tube (or thin-walled PCR tube if using a thermal cycler):

Table 2

Component	Volume (μl)
10X PCR buffer	2.5
25 mM MgCl₂	2.5
10 mM dNTP mix	1
0.1 M DTT	2.5
Final Volume	8.5

2) Incubate the mixture 10 min at 70°C to denature RNA. Chill 1 min on ice. Collect the contents of the tube by brief centrifugation and add the following in the order given:

3) Mix gently, and collect the reaction by brief centrifugation. Incubate for 1 min at 42°C.

4) Add 1 μl of SuperScript™ II RT. Mix gently and incubate for 50 min at 42°C.

5) Incubate at 70°C for 15 min to terminate the reaction.

6) Centrifuge 10 to 20 s and place the reaction at 37°C.

7) Add 1 μl of RNase mix, mix gently but thoroughly, and incubate for 30 min at 37°C.

8) Collect the reaction by brief centrifugation and place on ice. The procedure may be stopped at this point and the reactions stored at -20°C.

4. S.N.A.P. Column Purification of cDNA

1) Equilibrate the binding solution to room temperature.

2) For each sample to be purified, equilibrate ~100 μl of sterilized, distilled water at 65°C for use in step 9.

3) Add 120 μl of binding solution (6 M NaI) to the first strand reaction.

4) Transfer the cDNA/NaI solution to a S.N.A.P. column. Centrifuge at 13,000 x g for 20 s.

5) Remove the cartridge insert from the tube and transfer the flowthrough to a microcentrifuge tube. Save the solution until recovery of the cDNA is confirmed. Place the cartridge insert back into the tube.

6) Add 0.4 ml of COLD (4°C) 1X wash buffer to the spin cartridge. Centrifuge at 13,000 x g for 20 s. Discard the flowthrough. Repeat this wash step three additional times.

7) Wash the cartridge two times with 400 μl of COLD (4°C) 70% ethanol as described in step 6.

8) After removing the final 70% ethanol wash from the tube, centrifuge at 13,000 x g for 1 min.

9) Transfer the spin cartridge insert into a fresh sample recovery tube. Add 50 μl of sterilized, distilled, water (preheated to 65°C) to the spin cartridge. Centrifuge at 13,000 x g for 20 s to elute the cDNA.

5. TdT Tailing of cDNA

1) Add the following components to each tube and mix gently:

Table 3

Component	Volume (μl)
DEPC-treated water	6.5
^{5X tailing buffer}	5.0
2 mM dCTP	2.5
S.N.A.P.-purified cDNA sample	10.0
Final Volume	24.0
Component	Volume (μl)

2) Incubate for 2 to 3 min at 94°C. Chill 1 min on ice. Collect the contents of the tube by brief centrifugation and place on ice.

3) Add 1 μl TdT, mix gently, and incubate for 10 min at 37°C.

4) Heat inactivate the TdT for 10 min at 65°C. Collect the contents of the reaction by brief centrifugation and place on ice.

6. PCR of dC-tailed cDNA

1) Equilibrate the thermal cycler block to 94°C. In most cases, the “good start” procedure gives specific amplification products. For some target and primer sets, “hot start” has been reported to improve the specificity of the reaction.

2) Add the following to a 0.2 or 0.5-ml thin-wall PCR tube sitting on ice:

Table 4

Component	Volume (μl)
sterilized, distilled water	31.5
^{10X PCR buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl]}	5.0
25 mM MgCl₂	3.0
10 mM dNTP mix	1.0
nested GSP2 (prepared as 10 μM solution)	2.0
Abridged Anchor Primer (10 μM)	2.0
dC-tailed cDNA	5.0
Final Volume	49.5

3) Add 0.5 μl of Taq DNA polymerase (5 units/μl) immediately before mixing.

4) Mix the contents of the tube (Taq DNA polymerase is added immediately before going into the thermal cycler) and overlay with 50 to 100 μl of mineral oil (if necessary).

5) Transfer tubes directly from ice to the thermal cycler preequilibrated to the initial denaturation temperature (94°C).

6) Perform 30 to 35 cycles of PCR. A typical cycling protocol for cDNA with ≤1 kb amplified region is:

PAD: 94°C for 1-2 min

Cycle:

Denaturation: 94°C for 0.5-1 min

Annealing of primers: 55°C for 0.5-1 min

Primer extension: 72°C for 1-2 min

Followed by:

Final extension: 72°C, 5-7 min

Indefinite hold: 5°C, until samples are removed.

7) Analyze 5-20 μl of 5' RACE products by agarose gel electrophoresis according to standard protocols, using appropriate size standards (19). Either TAE or TBE electrophoresis buffer may be used for the procedure. The volume of the sample used for analysis will depend on the volume and thickness of the sample well. If products will be extracted for reamplification, ultraviolet (UV) visualization ofethidium bromide-stained products should be performed using either a long wavelength (356-nm) UV or 302-nm wavelength source to minimize DNA nicking.

7. Nested Amplification

1) Dilute a 5 μl aliquot of the primary PCR into 495 μl TE buffer [10 mM Tris-HCl, (pH 8.0), 1 mM EDTA].

2) Equilibrate the thermal cycler block to 94°C.

3) Add the following to a 0.2 or 0.5-ml thin-wall PCR tube sitting on ice.

Table 5

Component	Volume (μl)
sterilized, distilled water	33.5
^{10X PCR buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl]}	5.0
25 mM MgCl₂	3.0
10 mM dNTP mix	1.0
nested GSP (prepared as 10 μM solution)	1.0
AUAP or UAP (10 μM)	1.0
dilution of primary PCR product	5.0
Final Volume	49.5

4) Add 0.5 μl of Taq DNA polymerase (5 units/μl) immediately before mixing.

5) Mix the contents of the tube (Taq DNA polymerase is added immediately before going into the thermal cycler) and overlay with 50 to 100 μl of mineral oil (if necessary).

6) Transfer tubes directly from ice to the thermal cycler preequilibrated to the initial denaturation temperature.

7) Perform 30 to 35 cycles of PCR.

8) Analyze 5 to 20 μl of the amplified sample, using agarose gel electrophoresis, ethidium bromide staining, and the appropriate molecular size standards.