Paris Autism Research International Sibpair study

Primers used for the study of the glutamate 6 receptor (GluR6 or GRIK2) gene

Jamain S, Betancur C, Quach H, Philippe A, Fellous M, Giros B, Gillberg C, Leboyer M, Bourgeron T, and the Paris Autism Research International Sibpair (PARIS) Study. Linkage and association of the glutamate receptor 6 with autism. Molecular Psychiatry 2002, 7(3): 302-310.   PubMed

Exon	Exon size (bp)	PCR size (bp)	Primer name	Primer sequence
Exon 1	115	223	G6EX1F	5'-GGAGCCGAACGCTAGATCGGG-3'
			G6EX1R	5'-CCGGCTGCCTGGGAGCGGATAC-3'
Exon 2	168	274	G6EX2F	5'-TGTGAAAATTATGTTTATTCTTG-3'
			G6EX2R	5'-AGAAATTTGAATTCCAGGAAAC-3'
Exon 3	258	392	G6EX3F	5'-GGTATCTGTTGGCACACTGGCAC-3'
			G6EX3R	5'-AGAACCTGGAATGGGCTTCAG-3'
Exon 4	182	426	G6EX4F	5'-TTATGCGTTCTTGGCCGTATC-3'
			G6EX4R	5'-AGATCATATTGTAAGTGCAAG-3'
Exon 5	54	411	G6EX5F	5'-AAGCCATAGCTTTACAATTGCA-3'
			G6EX5R	5'-TAACTACAAAGGAATCAGTC-3'
Exon 6	174	313	G6EX6F	5'-GCCTGTGAAGATACTCTGTCC-3'
			G6EX6R	5'-ACCCAACTGAGGCCTTTGCTC-3'
Exon 7	144	270	G6EX7F	5'-TTGCAAACCATCTACCACAAGTT-3'
			G6EX7R	5'-CACTTCATTCAGCTTGACAACAA-3'
Exon 8	108	276	G6EX8F	5'-TATAAGTTTCCCTACTTTTGT-3'
			G6EX8R	5'-ATGAAATGTGACTACAACAGAG-3'
Exon 9	114	242	G6EX9F	5'-TGCAGTACTCTCTACCACGTGC-3'
			G6EX9R	5'-TGTCCTTTTCTATGCACTTCTGTG-3'
Exon 10	211	325	G6EX10F	5'-TATTCTGATGATGAGTTTCATG-3'
			G6EX10R	5'-GAGTAAACCTTGTAGCAACAT-3'
Exon 11	220	469	G6EX11F	5'-CTTTACTTCAGAGTAAGGAAT-3'
			G6EX11R	5'-GATTGGACTTTTGACAATTATTAG-3'
Exon 12	119	309	G6EX12F	5'-TTGTGCCAGCAAGAGCTGTCACCC-3'
			G6EX12R	5'-GCTTTCTACTTAAACTTATCTGTC-3'
Exon 13	217	368	G6EX13F	5'-TGATTCCATTCTGCCACTGTGAC-3'
			G6EX13R	5'-TCGAGAGAAGTTTGCGCTCTA-3'
Exon 14	227	410	G6EX14F	5'-TGTAGTTCATTGTGTCTAAGC-3'
			G6EX14R	5'-TTCCTAATGGCATACGTGGAC-3'
Exon 15	251	393	G6EX15F	5'-CCTCCTCTCATCTTGCTAACC-3'
			G6EX15R	5'-GGTTTGTGTTTTATTGTAACA-3'
Exon 15bis	86	350	G6EX15bF	5'-TGTTTCAAGAATTAGAGATGT-3'
			G6EX15bR	5'-CTCCACATTAATATAACAGAC-3'
Exon 15ter	145	251	G6EX15tF	5'-ACGGGATCAAGTTTTGACCAT-3'
			G6EX15tR	5'-TGTGGCACCTGACAGCCAGTA-3'
Exon 16	165	335	G6EX16F	5'-TGTGACAAGTCTGTTGAGGATGA-3'
			G6EX16R	5'-TTCCACAGGAAACATTCTGGC-3'
ECS		273	G6ECSF	5'-TGGATACATGAAAGAGGAACCC-3'
			G6ECSR	5'-TGAAACACTAAATACTCCCAAG-3'
RT PCRs			G6cd15	5'-GGAGAATTTTTATACAAATCCAA-3'
			G6cd15b	5'-TACCATCCAGACACTGTTTAG-3'
			G6cd15t	5'-AAGACTAAGTTACCTCAAGAC-3'
			G6cd16	5'-TTATGCCATGGTTTCTTTACCTGG-3'

Method for genotyping the two SNPs in intron 14 and exon 15 of the GluR6 (GRIK2) gene

The intron 14 C/T SNP and the exon 15 G/A SNP (E808E) can be genotyped in a single PCR amplification, using primers G6EX15F (5'-CCTCCTCTCATCTTGCTAACC-3') and G6HindIII (5'-CCCCCAGGGCACTGGCCTCTAAGCT-3').

Genotyping of the intron 14 C/T SNP is performed by digestion with EcoNI, which cleaves the C allele and generates two fragments of 73 and 143 bp out of the 216 bp PCR product.

Genotyping of the exon 15 G/A SNP (E808E) is performed by digestion with HindIII, through PCR-based mutagenesis. A mutagenising reverse primer (G6HindIII, shown above, with the desired substitution indicated in bold) is used to alter TT to AA, 4 bp downstream of the polymorphism site. PCR amplification with this primer introduces a new HindIII RFLP associated with the SNP. HindIII cleaves only the A allele and generates two fragments of 191 and 25 bp out of the 216 bp PCR product.