The document discusses the development of a rapid multiplex PCR assay for genotyping ABO and Rh-antigen blood types from buccal swab DNA, including detection of a ccr5 gene deletion associated with HIV resistance. The assay was verified with a 100% accuracy rate using known blood group antigen samples. This research tool may enhance bone marrow donor identification and contribute to public health research.

1. ABSTRACT
Introduction: For optimal outcomes when matching bone marrow donors
with their recipients, it is preferable to use bone marrow of identical or
compatible blood types. Bone marrow registries thus require high
resolution HLA genotyping data to match donor specimens with their
recipients. We developed a research assay to aid in these investigations,
which utilizes buccal swab DNA from potential donors to determine the
ABO and Rh-antigen genotypes. In addition, the assay detects a 32 bp
deletion in the CCR5 gene. Homozygous carriers of this deletion are
resistant to HIV-1 infection, and thus could be valuable stem cell donors
for HIV-infected recipients. Methods: This research assay consists of a
multiplex-PCR reaction with 5 fluorescently-labeled and 12 allele-specific
primers followed by capillary
electrophoresis on the new Applied Biosystems SeqStudio™ Genetic
Analyzer. Four targeted SNPs of the human ABO gene allow the
determination of the A, A2, B, O1,2 and O3 alleles. Rh-antigen
genotyping is determined by targeting a small deletion that differs
between the RhD and RhCE genes. The peak pattern is analyzed with
GeneMapper™ software, and the resulting peak/genotype table is
translated into a genotype/phenotype report using a standard
spreadsheet. Results: We verified this research assay by analyzing a
panel of DNA samples with known blood group antigens and CCR5 gene
types. The results of this verification were found to be 100% accurate.
Conclusions: This easy to use, rapid research assay may prove useful
for future development of bone marrow donor identification assays, and
other areas of public health studies. For Research Use Only. Not for use
in diagnostic procedures.
INTRODUCTION
Four SNPs in the human ABO gene are sufficient to genotype
the five major blood types A1, A2, B, O1, O2, O3
The four SNPs are circled.
MATERIALS AND METHODS
Allele-specific PCR is used for genotyping the SNPs
Required for each SNP: 1 locus-specific primer with 5’ fluorescent
label and 2 allele-specific (unlabeled) primers of different sizes.
Size separation and detection of fluorescently labeled allele-
specific amplicons by capillary electrophoresis (CE)
The capillary electrophoresis (CE) for PCR fragment analysis can
be quickly performed on the new Applied Biosystems SeqStudio™
Genetic Analyzer featuring an innovative and easy to use
cartridge-based CE system with fully integrated capillary array
(n=4), buffer and polymer.
Details of the position of locus and allele-specific PCR primers in
exons 6 and 7 of the human ABO gene
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Edgar Schreiber^, Scott Higdon^, Thoa Nong*, Jar-How Lee*
^Thermo Fisher Scientific, Genetic Sciences Division, South San Francisco CA USA
* Thermo Fisher Scientific, One Lambda TDX Division, Canoga Park CA USA
Concurrent Determination of ABO RhD Blood Types and the HIV-1 Resistance
Marker CCR5 Deletion Via a Rapid Multiplex PCR and Capillary Electrophoresis-
Based Genotyping Research Assay.
The RhD and CE genes are facing each other separated by an
unrelated gene segment in-between. The RhD and CE genes are
almost identical in nucleotide sequence however a small deletion is
present in an intron of the RhD gene which can be exploited for
genotyping: primers flanking the deletion will amplify the gene
segment from both genes if both genes are present. If the RhD gene
is deleted on both chromosomes only the RhCE gene segment will
amplify.
Distinguishing the RhD gene from the RhCE gene
The RhCE fragment can be used as a control/reference for the
copy number in determination of RhD+.
Heterozygous RhD deletion results in approximately half peak
height compared to homozygous state.
If the RhD gene is deleted on both chromosomes only the RhCE
gene segment will amplify leading to absence of a peak for RhD
and presence of a peak for Rh CE.
No D peak Only Rh CE peak
Detecting the 32 bp deletion in the CCR 5 gene
C chemokine receptor type 5, also known as CCR5 or CD195, is
a protein on the surface of white blood cells that is involved in
the immune system as it acts as a receptor for chemokines. This
is the process by which T cells are attracted to specific tissue
and organ targets. Many forms of HIV, the virus that causes
AIDS, initially use CCR5 to enter and infect host cells. Certain
individuals carry a mutation known as CCR5-∆32 in the CCR5
gene, protecting them against these strains of HIV.
https://en.wikipedia.org/wiki/CCR5
The deletion can be detected by fluorescent PCR using primers
flanking the deletion. The resulting amplicons are resolved by
CE and the deletion presents as a shorter fragment
Various off the shelf PCR master mixes
were tested; the Platinum™ PCR Master
Mix (Applied Biosystems) performed best
for this particular multiplex assay.
The Platinum Multiplex PCR Master Mix is
designed specifically for endpoint
multiplex PCR. It supports easy
multiplexing with minimal optimization.
Up to 20 amplicons can be amplified in a
single reaction.
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1 ul of the finished PCR reaction is
combined with a mix of 10 ul HiDi™
Formamide and 0.3 ul GeneScan™ 600
LIZ™ dye size standard for CE
electrophoresis on an Applied Biosystems
SeqStudio or 3500 Genetic Analyzer. The
resulting fsa trace file is analyzed with
GeneMapper™ software and the
electropherogram is reviewed for correct
peak capture and labeling.
Lastly, a genotype table is produced listing
the alleles detected with name, size and
peak height and area which is exported as
a csv file.
To determine the biological phenotype, i.e. blood type, the
peak pattern or “raw genotype” table needs to be
translated
Shown above are the 4 SNPs that determine the basic ABO blood types
a SNP to Phenotype
matrix is created
and a unique numerical
value is assigned for
each given allele
By summing up these values for the
diploid homozygous and heterozygous
alleles a unique genotype score can be
established that reflects a given blood
genotype (GT blood type).
Using this algorithm, standard spreadsheet
software and a simple “if - then” function
logic, the genotype table can be readily
translated into a fully annotated output
format with phenotype information.