Analysis of Gene Expression in Striatal Tissue by Multiprobe RNase Protection Assay - Drugs of Abuse: Neurological Reviews and Protocols - page 181

Table 1
PCR Primer Sequences

Annealing

Gene Primer sequence temperature

Cyc 5 - GGGA ATTCCAGGATTCATGTGCCAG-3 61°C

5 - CCGGA TCCTGAGCTACAGAAGGAATGG-3

D1 5 - GGGAATT CAGCTAAGCTGGCACAAGGCAA-3 61°C

5 - GGC TGCAGAATGGCTGGGTCTCC-3

D2 5 - GGGAATT CGCAGCAGTCGAGCTTTCAGA-3 61°C

5 - GGCTGCA GCTCATCGTCTTAAGGGAGGT-3

D3 5 - GGGAATT CTCACTCGACAGAACAGCCA-3 63°C

5 - GGGGATCC GGACGTGGATAACCTGCCGT-3

“GC clamp” and added restriction endonuclease sites are noted in boldface.

8. PCR primer sequences may be designed with “GC clamps” at the 5 end to

facilitate annealing. It is convenient to include also restriction endonuclease
sites. Table 1 provides sets of PCR primers for amplifi cation of D1, D2, and
D3 dopamine receptors and cyclophilin. Each sequence is amplifi ed in a 20- L

PCR reaction solution containing 20 m M Tris-HCl, pH 8.3, 25 m M KCl, 1.5 m M
MgCl 2 , 0.2

µ

M each primer, 0.1 m M each dNTP, and 2 U of Ta q polymerase.
PCR reactions for D1 and D2 receptors and cyclophilin employed an initial
denaturation at 94°C for 1.5 min, followed by 30 cycles at 94°C for 30 s, 30 s
at 61°C, and 72°C for 30 s. The D3 receptor PCR reaction employed an anneal-
ing temperature of 63°C. Each reaction was terminated following a 3-min
elongation at 72°C. Amplifi ed PCR products were then separated by agarose gel
electrophoresis, and bands corresponding to the products of interest may be
purifi ed onto DEAE-cellulose membranes (5) . PCR amplifi cation across alterna-
tive splice site junctions provides products of different size, corresponding to each
splice isoform. To provide suffi cient product for subsequent steps, it is generally
necessary to reamplify the gel-purifi ed products by PCR under identical condi-
tions. The fi nal product may then be extracted once with phenol-CHCl 3 -isoamyl
alcohol (25

µ

24 1) and once with CHCl 3 -isoamyl alcohol (24 1), precipitated

with sodium acetate, and then washed with 70% ethanol.

9. Plasmids are constructed by resuspending the purifi ed PCR product in restriction
digest buffer. D1 and D2 receptor inserts were digested with Eco R I and Pst I,
and D3 and cyclophilin inserts digested with Eco R I and Bam H I. Following
purifi cation by agarose gel electrophoresis, restriction digest products are ligated
into the corresponding restriction sites of PGEM-4 with T4 DNA ligase according
to standard protocols ( [5] , Ligation Reactions, Ligation of Cohesive Termini
1.68). Plasmids are grown in bacterial culture, purified, and plasmid DNA
sequenced to confi rm the identity of the insert in each plasmid construct.