Mutation and methylation of hMLH1 in gastric carcinomas with microsatellite instability

Table 1 Primer information for long and short PCR for HMLH1

A. Primer pairs for long-distance PCR
Exons 1-4
MLH1-4F GCG.GCT.AAG.CTA.CAG.CTG.AAG.GAA.GAA.CGT.GAa
MLH1-4R GGC.GAG.ACA.GGA.TTA.CTC.TGA.GAC.CTA.GGC.CC
Product size-10.8 kb
Exons 5-10
MLH5-10F GCG.CCC.CTT.GGG.ATT.AGT.ATC.TAT.CTC.TCT.ACT.GG
MLH5-10R GCG.CTC.ATC.TCT.TTC.AAA.GAG.GAG.AGC.CTG
Product size-10.5 kb
Exons 11-13
MLH11-13F CGG.CTT.TTT.CTC.CCC.CTC.CCA.CTA.TCT.AAG.G
MLH11-13R GGG.TTA.GTA.AAG.GAA.GAG.GAG.CTT.GCC.C
Product size-8.7 kb
Exons 14-19
MLH14-19F GGT.GCT.TTG.GTC.AAT.GAA.GTG.GGG.TTG.GTA.G
MLH14-19R GCG.CGC.GTA.TGT.TGG.TAC.ACT.TTG.TAT.ATC.ACA.C
Product size = 10.5 kb
B. Primer pairs for short PCR
Exon	Clampb	Product size	Tmc	Primer sequence
1	5	258	64.13	GCA.CTT.CCG.TTG.AGC.ATC
	40			CCG.TTA.AGT.CGT.AGC.CCT
2	40	187	38.14	ATA.AAT.TAT.TTT.CTG.TTT
				CAT.CCT.GCT.ACT.TTG.AGG
3	40	237	32.22	GGA.AAA.TGA.GTA.ACA.TGA
	2			TGT.CAT.CAC.AGG.AGG.ATA
4	2	218	36.26	ACC.CAG.CAG.TGA.GTT.TT
	40			GCC.CAA.AAT.ACA.TTT.CAG
5	40	170	30.19	ATA.TTA.ATT.TGT.TAT.ATT
				CAA.TTT.ACT.CTC.CCA.TGT
6	40	228	35.58	TTT.CAA.GTA.CTT.CTA.TGA
				ACT.TTG.TAG.ACA.AAT.CTC
7		194	30.88	GAC.ATC.TAG.TGT.GTG.TTT
	40			CCC.CTT.TTT.TCT.TTT.CAT
8	5	213	42.21	GAC.AAT.AAA.TCC.TTG.TGT
	50			AAG.ATT.TTT.TTA.TAT.AGG
9	40	249	33.73	TTT.GAG.TTT.TGA.GTA.TTT
				TGG.GTG.TTT.CCT.GTG.AGT
10	50	240	41.47	CAC.CCC.TCA.GGA.CAG.TTT
				ACA.TCT.GTT.CCT.TGT.GAG
11.1	50	145	40.58	AGG.TAA.TTG.TTC.TCT.CTT
				GAA.GTG.AAC.TTC.ATG.CTT
11.2	40	224	60.81	TCC.CAA.GAA.TGT.GGA.TGT
	2			AAA.GGC.CCC.AGA.GAA.GTA
12.1	40	184	44.53	TTT.TTT.TTT.TTT.TAA.TAC.A
				AAT.CTG.TAC.GAA.CCA.TCT
12.2	8	366	53.23	TGG.AAG.TAG.TGA.TAA.GGT
	40			TGT.ACT.TTT.CCC.AAA.AGG
13	40	272	49.06	ATC.TGC.ACT.TCC.TTT.TCT
				AAA.ACC.TTG.GCA.GTT.GAG
14	45	235	48.94	TAC.TTA.CCT.GTT.TTT.TGG
	5			GTA.GTA.GCT.CTG.CTT.GTT
15	40	179	29.97	CAG.CTT.TTC.CTT.AAA.GTC
				CAG.TTG.AAA.TGT.CAG.AAG
16		261	47.56	CTT.GCT.CCT.TCA.TGT.TCT.TG
	40			AGA.AGT.ATA.AGA.ATG.GCT.GTC
17	40	199	47.01	ATT.ATT.TCT.TGT.TCC.CTT
				AAT.GCT.TAG.TAT.CTG.CCT
18	45	215	46.67	CCT.ATT.TTG.AGG.TAT.TGA.AT
				GCC.AGT.GTG.CAT.CAC.CA
19		282	43.43	TGT.TGG.GAT.GCA.AAC.AGG
	40			ATC.CCA.CAG.TGC.ATA.AAT

^aUnderlined nucleotides represented nucleotides added to modify the melting temperatures of the primers.

^bGClampsm are: 50 clamp,CGC.CCG.CCG.CCG.CCC.GCC.GCG.CCC.CGC.GCC.CGT.CCC.GCC.GCC.CCC.GCC.CG; 45 GC clamp, CGC.CCG.CCG.CGC.CCC.CGC.CCC.GTC.CCG.CCG.CCC.CCG.CCC.GGC.CCG; 40 clamp, CGC.CCGCCG.CGC.CCC.GCG.CCC.GGC.CCG.CCG.CCC.CCG.CCC.G; 8 clamp, CGT.CCC.GC; 5 clamp, GCG.CG; 2 clamp,CG;

^cTM is given in % UF.

Table 2 Multiplex groups for short PCR

Multipex group I		Multipex group II
Exon	Final concentration	Exon	Final concentration
11.1	0.375 µM	5	1.5 µM
15	0.5 µM	2	1.25 µM
12.1	0.375 µM	7	1.75 µM
17	0.5 µM	4	1.625 µM
8	0.375 µM	11.2	0.5 µM
18	1.25 µM	6	1.625 µM
14	1.25 µM	9	1.5 µM
3	0.625 µM	1	1.25 µM
10	0.625 µM	13	1.625 µM
16	1.0 µM	12.2	0.325 µM
19	1.75 µM

Table 3 The relevance of MSI and hMLH1 mutation

MSI Status	No.of cases	hMLH1 mutation
MSI-H	8	3
MSI-L	9	0
MSS	51	0

Table 4 The relevance of methylation of hMLH1 and MSI

MSI status	Unmethylated	Hypermethylation(%)
MSI-H	1	7 (87.5)
MSI-L	8	1 (11.1)b
MSS	48	3 (5.9)d

^bP < 0.01;

^dP < 0.001 vs MSI-H group.