Nlaiii

Genomic DNA was purified individually from human myeloid and lymphoid cells engrafted in mice. LAM-PCR analysis was performed as described previously.^{17 (link)} Vector LTR junction sequences were amplified by linear PCR using 300 ng of genomic DNA, vector LTR–specific 5′ biotinylated primer, 5′-AGAACCTTGTGTCTCTCATCCC-3′, and PCR master mix. DNA was denatured at 94 °C for 5 minutes, followed by 50 cycles of amplification (94 °C for 45 seconds, 60 °C for 45 seconds, and 72 °C for 60 seconds), and a final extension at 72 °C for 10 minutes. Biotinylated PCR products were bound to streptavidin-conjugated magnetic beads overnight with mild shaking. After washing, double-stranded DNA was synthesized using a random hexamer (Invitrogen) and DNA polymerase I large fragment (NEB, Ipswich, MA). DNA was digested with MluCI, MseI, and NlaIII (NEB) and ligated to restriction enzyme-specific double-stranded linkers using the Quick Ligation Kit (NEB). DNA was denatured with 0.1 N NaOH and was PCR-amplified using LTR- and linker-specific primers (first nested PCR primer of LTR, 5′-ACCTCCTTCCCTGTAATACTC-3′, and primer of linker, 5′-GCACTCGTGCTCGACTGATAC-3′; second nested PCR primer of LTR, 5′-CCTGGTTTCTAGTGGCATTC-3′ and primer of linker, 5′-CCGTCGTATCGTAGCACAG-3′). A schematic diagram shows the LAM-PCR analysis (see Supplementary Figure S4).

Standard EDTA tubes were used to obtain blood samples, and DNA was obtained from each patient’s white blood cells using the QIAmp^® DNA Blood Mini Kit. The blood samples were collected from patients during clinical evaluation. The DNA was extracted and analyzed by polymerase chain reaction (PCR) assay during the patient’s evaluation. After restriction fragment length polymorphism processing, IL-6 −174G/C polymorphism was analyzed using PCR (Figure 1). The primers for polymorphism analysis was used with the standard primers. Digest 164 bp products of PCR was completed at the condition of 5 units NlaIII (New England Biolabs) at 37°C for 4 hours. Restriction fragments were analyzed using 3% ethidium bromide-stained agarose gel. The 111- and 52-position fragments are consistent with the homozygous genotype C/C; 164 bp represents the homozygous genotype G/G. The three fragments in the positions of 111, 52, and 164 bp represent the heterozygous genotype G/C. The reliability of samples was analyzed and evaluated. A negative control was used to prevent false positive results.