Amplification of 3'-end cDNA by classical RACE

Summary

This experiment describes the amplification of 3'-end cDNA by classical RACE.PCR Lab Guide, Section 25.4, Protocol 1

Operation method

Amplification of 3'-end cDNA by classical RACE

Materials and Instruments

dNTP solution Dithiothreitol TE Reverse transcription buffer RNA enzyme H RNasin SuperScript II reverse transcriptase poly(A)+RNA or total RNA QT primer Hercules Hot-Start polymerase
Water Bath or Heater Thermocycler

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reagents

The materials needed for this procedure can be purchased from most major suppliers along with the appropriate 5X enzyme reaction buffer or 10X enzyme reaction buffer.Superscript II reverse transcriptase and RNAase H can be purchased from Invitrogen, RNasin can be purchased from Promega Biotech, and Hercules thermostable Hot- StartDNA polymerase can be purchased from Stratagene, Tdt can be purchased from Invitrogen or BoehringerMannheim. StartDNA polymerase is available from Stratagene, and Tdt is available from Invitrogen or BoehringerMannheim. Use the enzymes according to the supplier's instructions. There are a number of different heat-stable DNA polymerases, any one of which is available, and the researcher should look for the most efficient amplification (with reference to reliability or price). The program from BoehringerMannheim is another good choice. The oligonucleotide primer sequences are listed in the notes to Figure 25-2, and can be used "unprocessed" except for the _QTs, which need to be purified to ensure that they are all full-length sequences (a service offered by most primer synthesis companies). 100 mmol/L dNTP solution is available from many suppliers.

This protocol is divided into two phases.

Phase 1: Reverse transcription to generate the cDNA template
Phase 2: Amplification

Phase 1: Reverse transcription to produce cDNA template

I. Materials

1. Buffers, solutions and reagents

dNTP solution (containing 4 dNTP, 10 mmol/L each)

Dithiothreitol (DTT) (0.1 mol/L)

TE (10 mmol/L Tris-HCl, pH 7.5, 1 mmol/L LTA, pH 8.0)

2. Enzyme and buffer

Reverse transcription buffer, 5X (supplied by manufacturer)

RNAase H

RNasin

SuperScript II reverse transcription (Invitrogen)

3. Nucleic acids and oligonucleotides

poly(A)+RNA or total RNA

_QT primer (100ng/ul)

4. Specialized equipment

Water bath or heater preset to 37°C, 42°C, 50°C, 70°C, 80°C

II. Methods

1. In a sterile centrifuge tube, mix the following transcription components on an ice bath.

Reverse transcription buffer, 5X 4ul

dNTP solution (10 mmol/L) 1ul

DTT (0.lmol/L) 2ul

Qt primer (100ng/pl) 0.5ul

RNasin(40U/ul) 0.25ul

2. In another tube, add 1ul of Ply(A ^)+RNA or 5ug of total RNA to 13ul of water, incubate at 80°C for 3 min, cool rapidly on ice, and centrifuge for 5s.
Ply(A ^)+RNA is preferred for reverse transcription to reduce the background of the reaction, but it is not necessary to prepare poly(A ^)+RNA if only total RNA is available.

3. Add RNA to the reverse transcription fraction, then add 1ul (200U) of Superscript II Reverse Transcriptase and incubate at room temperature for 5 min, 42°C for 1h, and 50°C for 10 min.

4. Incubate at 70°C for 15 min to inactivate the reverse transcriptase and centrifuge for 5s.

5. Add 0.75ul (1.5U) of RNAase H and incubate at 37°C for 20 min to digest the RNA template.

6. Dilute the reaction mixture to 1ml with TE and store at 4°C (this is the 3' end cDNA library).



Phase 2: Amplification

i. Materials

1. buffers, solutions and reagents

dNTP solution (contains 4 dNTP, 10 mmol/L each)

TE (10 mmol/L Tris-HCl, pH 7.5, lmmol/LDTA, pH 8.0)

2. Enzyme and buffer

Hercules Hot-Start polymerase (Stratagene)

Hercules Hot-Start Polymerase Buffer (10X)

3. Nucleic acids and oligonucleotides

The 3' end of the cDNA library obtained from stage 1

Oligonucleotide primers _Q0, _Q1, GSP1 and GSP2 (25pmol/L) (see Figure 25-2 for _Q0 and _Q1 ).

4. Specialized equipment

Programmable Thermal Cycler

II. Methods

1. First round

(1) In a sterile 0.2 ml centrifuge tube, mix the following components.

Hercules Hot-Start Polymerase Buffer (10X) 5ul

dNTP solution (10 mmol/L) 1.0ul

Hercules Hot-Start Polymerase 2.5U

_H2O to 50ul

(2) Add 1ul of 3' end cDNA library (from Step 6 of Phase 1), 25pmol each of GSP1 and _{Q0 primers} .

(3) Mix well and heat on a DNA thermal cycler at 98°C for 5 min to denature the first strand product and activate the polymerase. Cool to a suitable temperature (56-68°C), anneal for 2 min, and extend at 72°C for 40 min.

(4) Perform 30 rounds of amplification cycles according to the following program.



2. Second round

(5) Dilute part of the amplification product of the first strand with TE at 1:20.

(6) Amplify 1ul of the above diluted material with the _Q1 primer and the GSP2 primer using the above procedure but omitting the 2 min annealing and the 40 min extension step at 72°C.

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