Paris Autism Research International Sibpair study

Primers used for the study of the neuroligin genes NLGN3 and NLGN4

Jamain S, Quach H, Betancur C, Råstam M, Colineaux C, Gillberg IC, Soderstrom H, Giros B, Leboyer M, Gillberg C, Bourgeron T, and the Paris Autism Research International Sibpair Study. Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism. Nature Genetics 2003, 34(1): 27-29.   PubMed

 Primers NLGN4 and NLGN4Y

Exon	Forward primer	Reverse primer	Sequencing
1X	NLGNXE1F	NLGNXYE1R	NLGNXE1R
1Y	NLGNYE1F	NLGNXYE1R	NLGNYE1R
2X (5')	NLGNXE2F2	NLGNXYE2R2	NLGNXYE2R2
2Y (5')	NLGNYE2F2	NLGNXYE2R2	NLGNXYE2R2
2	NLGNXYE2F	NLGNXE2Rbis	NLGNXYE2F1/NLGNXY2
3	NLGNXYE3F	NLGNXYE3R	NLGNXYE3R/NLGNXY8
4X	NLGNXYE4F	NLGNXYE4R	NLGNXYE4R
4Y	NLGNYE4F	NLGNXYE4R	NLGNXYE4R
5	NLGNXYE5dF	NLGNXYE5dR	NLGNXYE5F3/NLGNXYE5R3
6	NLGNXYE6Fbis	NLGNXYE6dR	NLGNXYE6F3/NLGNXYE6R3

 Primers cDNA

NLGN1	NLGN1spliceF	NLGN1spliceR
NLGN2	NLGN2spliceF	NLGN2spliceR
NLGN3	NLGN3spliceF	NLGN3spliceR
NLGN4/5	NLGNXY3	NLGNXY4
G3PDH	G3PDHF	G3PDHR

 Primer sequences NLGN4 and NLGN4Y

Exon	Primer	Sequence (5'-3')
1	NLGNXE1F	ATTCTTTAAGAAAACTGTCAGC
	NLGNYE1F	GGGGTGCTTCTTTTGGGAGGCT
	NLGNXYE1R	CACGGGAAAGGGGTGCATGGA
2	NLGNXYE2F	GGATGTGGATGCAGATTTGAA
	NLGNXE2Rbis	GTATTGTTTTCTGTTCCAGTG
	NLGNXY2	TCATCCAGGTGCTGGGGGCAC
3	NLGNXYE3F	TGTGTTTCCGTACTTGGCTTT
	NLGNXYE3R	GCTTAGTCATTCACATGATGAA
4	NLGNXE4F	ACCAAAAATCTCTTGTGTTCT
	NLGNYE4F	AACAAAAATGTCCTGTGTTCT
	NLGNXYE4R	TTCTTGGTTCAGGGTATTTGC
5	NLGNXYE5dF	TGTCCRCAATTTTGCACCTGC
	NLGNXYE5dR	AGGAYAGTGATACCCCAACA
	NLGNXYE5F3	GGATAACGAGGACGGTGTGACGCC
	NLGNXYE5R3	TTCAGGGTAGCCGTAAAGGTTGTC
6	NLGNXYE6Fbis	AGAGCAGATTGTAACTTCCTG
	NLGNXYE6dR	GAGGGATAGGARGGGAAATAG
	NLGNXYE6F3	CTCTAAGGACCCTCACAAAACAGG
	NLGNXYE6R3	GACGGCAATGGTGACACTTAATTC

 Primer sequences cDNA

NLGN1spliceF	CCCAGAATATCATTGATGGCA
NLGN1spliceR	GCTGTTCCACTTTGAGCTATT
NLGN2spliceF	CCCGCCTGCCCGCAGAACCTG
NLGN2spliceR	ATGGCGGTGCCACTCTGGGCG
NLGN3spliceF	CCAGAACATCCACACAGCTGT
NLGN3spliceR	GCCACTTTGGATGATGGCTCT
NLGNXY3	TCCTGGACTGGCATCCGAAAT
NLGNXY4	GCTCTGAATGATGGCCTTCTG
G3PDHF	TGAAGGTCGGAGTCAACGGAT
G3PDHR	CATGTGGGCCATGAGGTCCAC

 Universal code: M(AC), R(AG), W(AT), S(CG), Y(CT), K(GT), V(ACG), H(ACT), D(AGT), B(CGT), N(ACGT)

PCR conditions NLGN3 gene

 PCR NLGN3 E1F-E1R (sequenced with E1F)

Cycles:	95°C	30 sec
	95°C	30 sec
	65°C - 55°C	20 sec	x 20
	72°C	30 sec
	95°C	30 sec
	55°C	10 sec	x 20
	72°C	30 sec
	72°C	10 min

 PCR NLGN3 E2F1-E2R1 non coding (sequenced with E2R1)

Cycles:	95°C	30 sec
	95°C	30 sec
	65°C - 50°C	20 sec	x 20
	72°C	30 sec
	95°C	30 sec
	50°C	10 sec	x 20
	72°C	30 sec
	72°C	10 min

 PCR NLGN3 E2F-E2R (sequenced with E2R)

Cycles:	95°C	30 sec
	95°C	30 sec
	65°C - 55°C	20 sec	x 20
	72°C	45 sec
	95°C	30 sec
	55°C	10 sec	x 20
	72°C	45 sec
	72°C	10 min

 PCR NLGN3 E3Fbis-E3Rbis (sequenced with E3Fbis)

Cycles:	95°C	30 sec
	95°C	30 sec
	57°C	20 sec	x 35
	72°C	30 sec
	72°C	10 min

 PCR NLGN3 E4F-E4R (sequenced with E4R)

Cycles:	95°C	30 sec
	95°C	30 sec
	57°C	20 sec	x 35
	72°C	30 sec
	72°C	10 min

 PCR NLGN3 E5F-E5R (sequenced with E5F1bis & E5R1)

Cycles:	95°C	30 sec
	95°C	30 sec
	57°C	20 sec	x 35
	72°C	1 min
	72°C	10 min

 PCR NLGN3 E6F-E6R (sequenced with HRNL3-1 & HRNL3-2)

Cycles:	95°C	30 sec
	95°C	30 sec
	57°C	20 sec	x 35
	72°C	1 min
	72°C	10 min

 Primers NLGN3

NLGN3E1F	TGGCACAAAAGGAGTTAATGG
NLGN3E1R	GATGGAGAACATGGGGTTGAG
NLGN3E2F	CCTATTGGGCTGATGCTGTGACC
NLGN3E2R	AGGGCACACAACCACATGCAAG
NLGN3E2F1	AGTGGATCATCTGTGGGATCA
NLGN3E2R1	GCTTCCCAAAGTGAGTGTTGA
NLGN3E2bisF	AGAAACAAGGAGATGGTGGGAC
NLGN3E2bisR	CTCAGATGAGGTGGCGCTTATG
NLGN3E2terF	GCCACACTGCAGTCATGCTGTT
NLGN3E2terR	GGCCAACAGGGCCAAAGACAGA
NLGN3E3Fbis	TTGAGCTCCAGGTTGAGCAACC
NLGN3E3Rbis	CCCCTTGCGAAGCCAGTCTTCC
NLGN3E4F	CTGCGTGCTCATTCTCTATTCC
NLGN3E4R	GTAGAAGAGAGCTGGCCGATTC
NLGN3E4F1(hplc)	TCCTGCGTCAGCCTCCTCACG
NLGN3E5F	ATGGCTATGTGTGACACGACAG
NLGN3E5R	GGAAGATGAGTGAAGGGGTACC
NLGN3E5F1bis	CCTGAGGATGGTGTCTCTGGCA
NLGN3E5R3	CAGTGGGGCCTACCATAGGAAC
NLGN3E5R2(hplc)	AGACAGTCCACCATATCCACG
NLGN3E6F	TTTCCTCATCCAGATAGAGTGG
NLGN3E6R	CATGTGTTCCTGGATCTGGGAG
NLGN3E6F1	GCTGGCAGCATTACAACTGGGC
NLGN3E5(CT)PvuII	CCGTGACAACCCTGAGACCAGC
NLGN3E6(AG)AluI	GTTGGGGATCATAGTGATGGAG
NLGN3E6R1(g/Ser)	TTGGGGATCATAGTGATGGTGC
NLGN3mutF	GACAACCCTGAGACCCGCTGTAAAACACTGGTGGC
NLGN3mutR	CTGTTGGGACTCTGGGCGACATTTTGTGACCACCG

 

PCR conditions NLGN4/4Y gene

 PCR NLGNY E1F-NLGNXY E1R (sequenced with E1R)

Cycles:	95°C	30 sec
	95°C	30 sec
	70°C - 60°C	20 sec	x 20
	72°C	30 sec
	95°C	30 sec
	55°C	10 sec	x 20
	72°C	30 sec
	72°C	10 min

 PCR NLGNX E1Fbis-NLGNXY E1R (sequenced with E1R)

Cycles:	95°C	30 sec
	95°C	30 sec
	65°C - 55°C	20 sec	x 20
	72°C	30 sec
	95°C	30 sec
	55°C	10 sec	x 20
	72°C	30 sec
	72°C	10 min

 PCR NLGNX E2F2-NLGNXYE2R2 & NLGNY E2F2-NLGNXYE2R2 (sequenced with E2R2)

Cycles:	95°C	30 sec
	95°C	30 sec
	50°C	15 sec	x 35
	72°C	30 sec
	72°C	10 min

 PCR NLGNXY E2F-E2Rbis (sequenced with E2F1 & NLGNXY2)

Cycles:	95°C	30 sec
	95°C	30 sec
	50°C	15 sec	x 35
	72°C	30 sec
	72°C	10 min

 PCR NLGNXY E3f-E3R (sequenced with E3R and sometimes with NLGNXY 8)

Cycles:	95°C	30 sec
	95°C	30 sec
	50°C	15 sec	x 35
	72°C	30 sec
	72°C	10 min

 PCR NLGNX E4F-NLGNXY E4R (sequenced with E4R)

Cycles:	95°C	30 sec
	95°C	30 sec
	50°C	15 sec	x 35
	72°C	30 sec
	72°C	10 min

 PCR NLGNY E4F-NLGNXY E4R (sequenced with E4R)

Cycles:	95°C	30 sec
	95°C	30 sec
	50°C	15 sec	x 35
	72°C	30 sec
	72°C	10 min

 PCR NLGNXY E5F-E5R (X) & NLGNXY E5dF-E5dR (Y) (sequenced with E5F3 & E5R3)

Cycles:	95°C	30 sec
	95°C	30 sec
	65°C - 55°C	20 sec	x 20
	72°C	1 min
	95°C	30 sec
	55°C	10 sec	x 20
	72°C	1 min
	72°C	10 min

 PCR NLGNXY E6Fbis-E6dR (sequenced with E6F3 & E6R3)

Cycles:	95°C	30 sec
	95°C	30 sec
	65°C - 55°C	20 sec	x 20
	72°C	1 min 15
	95°C	30 sec
	55°C	10 sec	x 20
	72°C	1 min 15
	72°C	10 min

 Primers NLGN4X/Y

NLGNXYE2bisdF	CTTTTWCTRGGTAGCATCTYTC
NLGNXYE2bisdR	ATAGATAGAYATAAAATCATGC
NLGNXYE2bisdF1	ACSAGTAATGACCRTGGTGAAG
NLGNXE4F1(hplc)	CTGGATCAGATTCAAGCACTGC
NLGNXE5R4(hplc)	CTTGTAGTTCTTGTTCCGCAGG
NLGNX(CT)E5NcoI	GCAGGCATTCTACCATGTCCAT
NLGNX(CT)E5SnaBI	GCAGGCATTCTACCATGTACGT
NLGNXY12	TTTACCCCACGGACATTCCACC
NLGNXY13	AACTCTATGAAGATTCTATAAC
NLGNXYE1aF	GAAACAAYGAATTTCCTCCAAA
NLGNXYE1aR	AGTGAGGCTTTCCMTCCTTTGC
NLGNXYE1bR1	GAAGCAAAACCACCAGCTCTAC
NLGNXYPF1	ACAGAGCTCTTATTCAGCCAC
NLGNXYPR1	GAGGATGTATTGAAAGCCCAGG
NLGN3PF	AACTGCCTGTTCTCACGTGAC
NLGN3PF1	CGTGCACAAGGAATGGAACTC
NLGN3PR	CTCCTGTGGCTCCTCAGAATG
NLGNXPF	TCCAACTTTCTCCAACTCAAC
NLGNYPF	GAAGCCAACTTTAAATACTGGG
NLGNYE1aF	TGCTGTGCGCATCTGGGTACC
NLGNXYPR	TCCACAGCTACAACCTCATGC
NLGNXE2F2	GCCACATCACTGGGACAGCTG
NLGNXE6R2	ACGATAAGGGTCTGCCGGGAT
NLGNXYE1bdF1	CAMGTGATTGATTAGACCTGGC
NLGNXYE1aF1	GTGTAAAAAGAACTCTTAACGG
NLGNXE1cF2	CCACTCTGACTAAAGAAGGTGGA
NLGNXE1cF3	CTGATGAAGGTGATATACAGGTAGC
NLGNXE1aF2	GTTTGCTGTACGCGTCTGGTGC
NLGN4E1bisF1	CTTGCAGTGAGCTGAGATCG
NLGN4E1bisF	CGCCTGTAGTCCCAGCTACT
NLGN4E1bisR	CACCATAGAATGAAAACGTTCC
NLGNXYE6dR	GAGGGATAGGARGGGAAATAG