Table 1.

Primers and PCR Conditions^a

Primer Name	Primer Sequence(5′→3′)	Start	End	Product Size(bp)	Overlap(bp)
Primers for amplifying large fragmentsb:
CSP12L1F	ACCATAATGCCTTCATTTTCCTAGAG	104262020
CSP12L1R	TAAACTATGCCCATCTTAGGACCTTC		104273050	11,030
CSP12L2F	AAAGTCCTGTTAACTTTGAACGTTTCTT	104266097
CSP12L2R	TTTATTATTATTACAAGGTGGCCAGTCA		104275541	9,444
Primers used for reamplification and sequencingc:
CSP12S1F	TCATTGCCTCAGCATAGATT	104262183
CSP12S1R	GCCCACCATTGAAAGACTAT		104262709	526
CSP12S2F	ACCACTATTGGGCTACCATT	104262415
CSP12S2R	GGTTTTCCCAATAACCTGAC		104262966	551	294
CSP12S3F	ATTTGGGGTCTCAAATGAAT	104262723
CSP12S3R	GTTTCCCTCTCTTCTCCAAA		104263363	640	243
CSP12S4F	AAAGTTTTCTGGGGCATAAC	104263089
CSP12S4R	AGCAACTTGGTCATCTTGAA		104263688	599	274
CSP12S5F	TTTGGAGAAGAGAGGGAAAC	104263344
CSP12S5R	ATTTGGCAAAGCTGATGTTA		104263874	530	344
CSP12S6F	CTCTGGGTTTGCAAGTAGTG	104263481
CSP12S6R	GATGCTGCCCTAAGGATAAT		104264167	686	393
CSP12S7F	TCCTATCAGGCTTCTCCTTC	104263827
CSP12S7R	GCAAGAGTCGATACATGAGG		104264515	688	340
CSP12S8F	ATTATCCTTAGGGCAGCATC	104264167
CSP12S8R	TCAGGAGAGATGCTAGTGGA		104264692	525	348
CSP12S9F	CCTCATGTATCGACTCTTGC	104264492
CSP12S9R	ACTCCCTTCCTTCCTTCTTT		104265052	560	200
CSP12S10F	ACTCAGCCTCCTCTCCTAAG	104264708
CSP12S10R	CCTTTCTTCCTTCCTTCCTT		104265213	505	344
CSP12S11F	AGGCCTAGCACACAATTACA	104264997
CSP12S11R	GGAGAACAGGAGCAATTTTT		104265678	681	216
CSP12S12F	TAGTCCCTCAGTGCTCACAT	104265439
CSP12S12R	AGCCAACCACTAAAACCATT		104265946	507	239
CSP12S13F	AAAAATTGCTCCTGTTCTCC	104265659
CSP12S13R	TTTCAAATCTTCCACACCAC		104266330	671	287
CSP12S14F	GGTTTTAGTGGTTGGCTTCT	104265930
CSP12S14R	TGCATATGTGGATGTTTGTG		104266580	650	400
CSP12S15F	AAGCAATGAAGTCCTTTTCC	104266329
CSP12S15R	ACTCAAGTGGGGTCTGTTTT		104266859	530	251
CSP12S16F	ACACACAAATGCACACACAT	104266624
CSP12S16R	AAAGACAAACCCAAGGTCAT		104267149	525	235
CSP12S17F	AACAGACCCCACTTGAGTTT	104266842
CSP12S17R	ACTCAAGGGTCTCTTTCAGG		104267360	518	307
CSP12S18F	ATGACCTTGGGTTTGTCTTT	104267130
CSP12S18R	TCTGCTGCTCCATAGTGAAT		104267755	625	230
CSP12S19F	TTGCCCAGTGGTTTTTAGTA	104267553
CSP12S19R	TTAATTGGCAGCTGAAAGAG		104268192	639	202
CSP12S20F	GGTGCAGAGCTTTTGTCTTA	104267935
CSP12S20R	GAGGGTGTATTTTCATGCAG		104268567	632	257
CSP12S21F	CTTTCAGCTGCCAATTAAGA	104268175
CSP12S21R	TCACAAAGGCCTTAAGATCA		104268751	576	392
CSP12S22F	GCCTCTCTTTCTCCATCACT	104268413
CSP12S22R	GCAGTAAGCAGTTTTGAGGA		104269035	622	338
CSP12S23F	CCTGATCTTAAGGCCTTTGT	104268730
CSP12S23R	GGAGATGTCTCAGAGAATGGT		104269343	613	305
CSP12S24F	AGGCTCTCATTCCTCAAAAC	104269006
CSP12S24R	AGACATGTTGGTCATGGAAG		104269511	505	337
CSP12S25F	AGTGCTCACAGCATGAACTT	104269168
CSP12S25R	AGAAGGTTTGTTGCCCTAAG		104269772	604	343
CSP12S26F	GAATTCTTCCATGACCAACA	104269487
CSP12S26R	GTGGGAAAAGAGGAAGAGAA		104270023	536	285
CSP12S27F	GGGCAACAAACCTTCTATTT	104269757
CSP12S27R	CTGGCATAGAAAAGCACAAC		104270408	651	266
CSP12S28F	TACCTGAGCTCTCAAATCCA	104269918
CSP12S28R	TGGGAAAGAGCATTGATAGA		104270442	524	490
CSP12S29F	TTTTGTATGCAATCCAATCC	104270190
CSP12S29R	ATGGCAATAGAGCTGATGAA		104270824	634	252
CSP12S30F	TTTGCCTATTCAACATCCAC	104270538
CSP12S30R	TTTCTTCCCTCCGTACTCTC		104271093	555	286
CSP12S31F	TGCCAAAACTAGGTCTCAAA	104270774
CSP12S31R	GCCCTGAGTAAGAACTTGGT		104271330	556	319
CSP12S32F	AGGGAGAATTGAGAGTACGG	104271064
CSP12S32R	GGGTTTTGTTTTTGCTTTTT		104271601	537	266
CSP12S33F	TTGGTAAAAGGGAGTACCAAG	104271296
CSP12S33R	CAGTGAGCCAGGATGTTTAG		104271864	568	305
CSP12S34F	CCTGCAACGTTTTATATTGC	104271596
CSP12S34R	ATAGGGAATTCATGGGTCAG		104272195	599	268
CSP12S35F	TCTGGAGTAGGAATCAGCAA	104271920
CSP12S35R	TCCCTCTGCTGAAATGTAGA		104272522	602	275
CSP12S36F	CTCTAACGTCCACTTTGTGC	104272307
CSP12S36R	ATTTTGCTTGCTGTTGTCAT		104272980	673	215
CSP12S37F	TACATTTCAGCAGAGGGAGA	104272499
CSP12S37R	TATTGTGGGGCTAACAGCTA		104273152	653	481
CSP12S38F	TGGTGAAACCCTGTGTCTAC	104272751
CSP12S38R	ATGGCATTTTTGATGATTTG		104273342	591	401
CSP12S39F	ATGACAACAGCAAGCAAAAT	104272961
CSP12S39R	ATTTGGGAACCACTACCCTA		104273480	519	381
CSP12S40F	ATATTTTGCCTGCAGTTTGA	104273196
CSP12S40R	TCCCTGAATCTATTTCACCA		104273697	501	284
CSP12S41F	TAGGGTAGTGGTTCCCAAAT	104273457
CSP12S41R	CTCCACATTTCTGCTCTCTG		104273981	524	240
CSP12S42F	GGAGAAGCTCCTGTCTTGTT	104273636
CSP12S42R	TTTATGGCTGTCCTTTGAGA		104274319	683	345
CSP12S43F	CATGTTGTAGCTGACCCATT	104273918
CSP12S43R	GAAAACACCTTCTGCTTCCT		104274563	645	401
CSP12S44F	GGTTTGCATTTTTAGTGCTG	104274180
CSP12S44R	ATGGCATCAGACAGACAAAC		104274702	522	383
CSP12S45F	GAAAAAGCTGTGAAAGCAAA	104274375
CSP12S45R	TGAGTGGATCAGGAAAGAGA		104274877	502	327
CSP12S46F	TCCTTTGGAAAATAGGAAGC	104274531
CSP12S46R	CCTTGCCATGTGAAATTAAA		104275049	518	346
CSP12S47F	TGGAAGTTAAGGGAAAGAGG	104274767
CSP12S47R	GTAGGGTAGGCATCTCTGCT		104275461	694	282

^aWe designed primers from a human DNA sequence (GenBank accession number NC_000011) and used the Expand 20kb^Plus PCR System (Roche) to amplify either a fragment spanning 11,030 bp (positions on chromosome 11: 104262020–104273050) or 9,444 bp (104266097–104275541). Using these PCR products as templates, we amplified overlapping fragments with a size of ∼500–700 bp and sequenced them with all of the nested primers. Sequence traces were processed by the program ExoTrace, developed at The Wellcome Trust Sanger Institute, and all potentially polymorphic positions flagged by the program were checked manually. Variable positions were compared in overlapping and complementary reads in all individuals. Primer sequences and PCR conditions are shown here.

^bPCR conditions for amplifying large fragments were according to manufacturers’ protocol: reaction in 25μl at 92°C for 2 min 15 s; followed by a cycle at 92°C for 10 s, at 60°C for 30 s, and at 68°C for 11 min 30 s 11 times; followed by a cycle at 92°C for 15 s, at 60°C for 30 s, and at 68°C for 11 min 30 s (increasing 10 s every cycle) 21 times; followed by a 68°C extension for 7 min.

^cPCR conditions for reamplification were as follows: PCR reaction in 15μl, 0.5μl of the template from the large-fragment PCR reaction (diluted 50 times), 0.5 U PlatinumTaq (Invitrogen), at 94°C for 6 min; followed by a cycle at 94°C for 45 s, at 60°C for 45 s, and at 72°C for 1 min 30 s 35 times; followed by a 3-min extension at 72°C.