Anchored-PCR

This procedure is based on that of Troutt et a l (1992) and Belyavsky et al. (1989). Specific mRNA sequences of the IL-5Ra were converted to cDNA by reverse transcription with the specific

oligonucleotide, CLIO, for anchored-PCR of the 5'-terminus and with CL12 [an oligo(dT)-containing adapter primer] for anchored-PCR of the 3'-terminus (refer to Table 3.2.1 and Figure 3.2.1 for sequences of and location of all primers on the cDNA). For the 5'-anchored-PCR, the reverse transcription mixture consisted of 3 jig total RNA from the BCLi(5Bib) cell line, 20 pmoles CLIO primer, 400 U Moloney murine leukemia virus reverse (MMLV-RT) transcriptase, 50 U RNasin, 0.1 m g /m l bovine serum albumin, 0.5 mM dNTPs, 500 mM Tris-HCl (pH 8.4), 75 mM KC1, 3 mM MgCl2 and 10 mM DTT, in a total volume of 50 jil which was incubated at 37°C for 1 hour. For the 3’-anchored- PCR, all components of the reverse transcription reaction were the same as that for the 5'-terminus, except 0.5 |ig of CL12 was used instead of CLIO.

After reverse transcription, excess primer was removed by selective precipitation using cetyltrimethyl ammonium bromide (CTAB). To the 50 pi reverse transcription reaction mixture, the following were added: 20 pi of 0.1 M N aC l/40 mM EDTA, 1 pi of

0.5 p g /m l Polyl.Poly C and 3 pi of 10% CTAB. Following centrifugation at 12 OOOxg for 20 minutes at room temperature, excess primer

remaining in the supernatant was removed. The pellet was dissolved

in 14 pi of 1 M NaCl and reprecipitated by adding 25 pi of H2O and 1 pi

of 10% CTAB. After centrifugation, the pellet was dissolved in 10 pi of 1 M NaCl and reprecipitated with 27 pi ethanol, centrifuged and dried. The pellet was resuspended in 20 pi of 50 mM N aO H /2 mM EDTA and heated at 65°C for 30 minutes to hydrolyse the RNA. The cDNA was concentrated by neutralising with 2 pi of 3 M sodium acetate (pH 5.2) and precipitated with 1 pi of 1 p g /p l glycogen and 70 pi of ethanol. After

1 hour at -20°C the mixture was centrifuged and the pellet dried.

The cDNA for 3’-anchored PCR was dissolved in 30 pi of water, but for 5'-anchored PCR, the anchor oligonucleotide, CL16, was ligated to the cDNA.

Prior to ligating CL16 to the cDNA, it has to be blocked at its 3'-end with dideoxyATP to prevent self-ligation. This was performed in a reaction mixture consisting of 1 nmol CL16, 14 nmol ddATP, 10 U terminal deoxynudeotidyltransferase, 100 mM potassium cacodylate,

2 mM C0CI2 and 0.2 mM DTT in 70 pi which was incubated at 37°C for

1 hour. After that the 5'-end of the modified CL16 was phosphorylated in a reaction mixture consisting of 50 pmol of modified C L 16,100 pmol ATP, 70 mM Tris-HCl (pH 7.6), 10 mM MgCl2/ 5 mM DTT, 5 U T4 polynucleotide kinase in 20 pi which was incubated at 37°C for 15 m inutes.

The ligation of the modified anchor oligonucleotide CL16 to the 5'-end of the cDNA was carried out in a 20 pi ligation mix containing

10 pmol of modified CL16, 50 mM Tris-HCl (pH 8), 10 mM MgCl2,

10 p g /m l bovine serum albumin, 10 mM hexamine cobalt chloride, 2 pM ATP, 25% polyethylene glycol 8 000 and 10 U T4 RNA ligase which was incubated overnight at 22°C. The ligation reaction was terminated by adding 32 pi of 0.5 M N aC l/10 mM Tris-HCl (pH 8)/lm M EDTA and the modified cDNA was precipitated by the addition of 1 pi of 1 p g / jil glycogen and 100 pi of ethanol. The pellet was collected by centrifugation and then dissolved in 30 pi of water.

In 5-anchored PCR, the first round of PCR was carried out using primers CL11 and the universal primer T3 (which anneals to CL16). After that 2 pi of the resultant PCR product was used for nested PCR using primers CL8 and T3 (see Figure 3.2.7.1). A PCR reaction mixture typically contained 20 pmol of each primer with the following cycling profile: 1 cycle at 94°C for 2 minutes; 35 cycles at 94°C for 30 seconds, 52°C for 30 seconds and 72°C for 1 minute; and 1 cycle at 72°C for 10 minutes in a Corbett FTS-1 thermal cycler. The PCR products were fractionated on a 1.5% agarose gel and gel slices for each band of PCR product centrifuged through a cone of filter paper (Whatman 541). The eluate was extracted with phenol /chloroform and the fragment made blunt ended with T4 DNA polymerase and then subcloned into the

Smal site of pBluescript SK(-). Recombinant clones were sequenced

using T3-dye primer fluorescence sequencing protocol (see Section 2.2.21).

In 3'-anchored PCR, the first round of PCR was carried out using primer pair CL13 and universal primer CL12. A 2 pi volume of the

5’

cDNA 1 --- --- AAAAAA

1

Convert mRNA to cDNA by reverse transcription

1

r Degrade RNA template by _{alkaline hydrolysis}

Ligate anchor oligonucleotide CL16 to cDNA

PCR with CL11 and universal primer T3

Nested PCR with CL8 and universal primer T3

CL12 mRNA

Y

Extend primer by reverse transcription

Y

TTTTT

CL12

Destroy RNA template by alkakine hydrolysis TTTTT < --- CL 12 PCR using CL13 and universal primer CL12 V CL 14

■TTTTT Nested PCR using CL14 and

universal primer CL12

Figure 3 2.7.2 Schematic representation of the 3'-anchored PCR

resultant PCR reaction mixture was used for nested PCR with primer pair CL14 and CL12 (see Figure 3.2.7.2). The PCR conditions and subcloning of the PCR product, if necessary, were similar to that described for 5'-anchored PCR.

3.3 Results