1. Development of HLA-B*57:01 Genotyping Real-Time PCR with Optimized Hydrolysis Probe Design 
Hou-Sung Jung, Gregory J. Tsongalis, Joel A. Lefferts 
The Journal of Molecular Diagnostics 
Volume 19, Issue 5, Pages (September 2017)
DOI: /j.jmoldx
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
DNA sequence polymorphisms between HLA-B*57:01 and HLA-B*57:03 and HLA-B*58:01-type alleles and location of primers and/or probes of HLA-B*57:01 genotyping real-time PCR. A: Exon 3 DNA sequences of HLA-B*57:01 were aligned with abacavir (ABC)-insensitive HLA-B alleles. HLA-B*57:11 and HLA-B*58:01 alleles represent HLA-B*58:01-type alleles, and HLA-B*57:02 and HLA-B*57:03 alleles do HLA-B*57:03-type alleles. Consensus nucleotides with those in HLA-B*57:01 are displayed with dashes. Polymorphic codons are underlined and their encoding amino acids are presented. If polymorphic codons are synonymous, amino acids are not presented. Numbers above the cDNA sequences are their positions from the start of cDNA sequences, and numbers under amino acid residues are their positions from the starting amino acid of protein sequences. Restriction enzyme recognition sites of NlaIII in HLA-B*57:01 and RsaI in HLA-B*57:02 and 57:03 are marked with plus signs and dots, respectively. B–D: Primer and probe positions are marked by solid and dashed boxes, respectively. Full primer/probe sequences are presented with closed boxes and partial ones are with open boxes. B: Primer location of sequence-specific primer (SSP) PCR.23–25 C: Primer location of real-time PCR with a double-stranded DNA binding fluorescence dye26 or with a hydrolysis probe.19 D: Primer and probe location designed and tested in the present study.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
Amplification graphs of the HLA-B*57:01 real-time PCR by the SmartCycler II (Cepheid, Sunnyvale, CA). A–C: Multiplex real-time PCR reactions were conducted with three probes of B5701-P2 [6-carboxyfluorescein (FAM)], B5703-P2 [tetrachlorofluorescein (TET)], and ACTA1-P1 (CAL Fluor Red 610) (Table 1). Twelve pre-genotyped genomic DNA were used as templates (Table 2). Fluorescence signals were detected from FAM (A), TET (B), and Texas Red (C) channels. D–F: gBlocks Gene fragments of HLA-B alleles with a single-nucleotide mismatch in primer or probe region were used as a template in real-time PCR. As controls, HLA-B*57:01 and HLA-B*57:02 fragments were also tested. The target copy numbers in the fragment template were calculated to match to those in the genomic DNA template. Fluorescence signals were obtained from FAM (D), TET (E), and Texas Red (F) channels.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
HLA-B*57:01 PCR–restriction fragment length polymorphism of pre-genotyped DNA samples. A: Multiplex PCR products were separated on 2% agarose gels. Upper bands were PCR products amplified by ACTA1-G2F and -G2R primers (Table 1). Products from HLA-B*57:01 and related alleles ran faster than the ACTA1 product on the 2% agarose gels. A 50-bp DNA ladder was used as a size marker. B and C: PCR products were digested with NlaIII (B) or RsaI (C) for 2 hours and then digested products were separated on 2% agarose gels. 1, NA02016; 2, NA12273; 3, NA17019; 4, NA17078; 5, NA17115; 6, NA17215; 7, NA17236; 8, NA17240; 9, NA17275; 10, NA17288; 11, NA17291; 12, NA Refer to Table 2 for detailed genotype information.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Sanger DNA sequencing results of sample A20 in exon 3 of HLA-B*57:01. A: Electropherograms of HLA-B exon 3 sequences from sample A20 are displayed. B: Sanger sequencing results of sample A20 are aligned with the exon 3 sequences of HLA-B*57:01. Consensus nucleotides are marked with vertical lines. Dots represent discordant nucleotides from HLA-B*57:01, and colons represent nucleotides matched to one of nucleotide signals in paired peaks.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions