MBL2 Genotypes and Their Associations with MBL Levels and NICU Morbidity in a Cohort of Greek Neonates

Table 1

PCR-RFLP protocol for the detection of MBL2 polymorphisms.

(a) PCR primers and conditions

Region	Position^*	Sequence^#	Conditions of both reactions	PCR product
Promoter	4421-4446; 4808-4830	5′-GAAAATGCTTACCCAG(G)ACAAGCCTGT-3′; 5′-GTCTCCTCATATCCCCAGGC-3′	94°C for 5 min, followed by 30 cycles (94°C for 30 s, 64°C for 30 s, and 72°C for 30 s) and a final elongation at 72°C for 5 min	134 bp
Exon 1	5191-5218; 5305-5324	5′-CATCAACGGCTTCCCAGG(C)G(A)CAAGATGGG-3′; 5′-GTCTCCTCATATCCCCAGGC-3′		410 bp

(b) Expected fragments of RFLP genotyping for MBL2 gene

Polymorphic sites	Enzyme	RFLP conditions	Wild-type homozygotes (bp)	Mutant homozygotes (bp)	Wild-type & mutant heterozygotes (bp)
-550G>C (rs11003125)	DrdI	4 h at 37°C	410 (H allele)	388 + 22 (L allele)	410 + 388 + 22 (H & L)
-221G>C (rs7096206)	BtgI	4 h at 37°C	410 (X allele)	352 + 58 (Y allele)	410 + 352 + 58 (X & Y)
Arg52Cys (rs5030737)	MwoI	12 h at 60°C	109 + 25	134	134 + 109 + 25
Gly54Asp	BanI	4 h at 37°C	99 + 35	134	134 + 99 + 35
Gly57Glu	MboII	4 h at 37°C	134	78 + 56	134 + 78 + 56

(c) MBL2 promoter genotypes: RFLP in MBL2 promoter using a combination of both DrdI and BtgI enzymes

Genotypes	Respective haplotypes^∧	410 bp	388 bp	352 bp	330 bp
PCR product		✓
HY/HY	HYA/HYA, HYA/HYD, HYD/HYD			✓
HY/LY	HYA/LYA, HYA/LYB, HYA/LYC, HYD/LYA, HYD/LYB, HYD/LYC			✓	✓
HY/LX	HYA/LXA, HYD/LXA		✓	✓
LX/LX	LXA/LXA		✓
LX/LY	LXA/LYA, LXA/LYB, LXA/LYC		✓		✓
LY/LY	LYA/LYA, LYA/LYB, LYA/LYC, LYB/LYB, LYB/LYC, LYC/LYC				✓

MBL2 gene numbering is according to GenBank accession number NG_008196.1.
^#In forward primers of both reactions, the nucleotides in parentheses were modified and changed to the underlined ones.
^∧The common haplotypes of MBL2, identified also in this study, are the HYA, LYA, LXA, HYD, LYB, and LYC.