BLOOD TYPING USING DNA

Abstract

Complex blood group typing can be performed at the DNA level, using for example, air-dried cheek swabs or finger prick blood in a microarray test that completely bypasses the need for DNA extraction prior to analysis of the blood group type.

Description

FIELD

The disclosure relates generally to blood typing. The disclosure relates specifically to blood typing using DNA.

BACKGROUND

Serology-based blood group typing is a progenitor of personalized medicine (2). Recently, the genetic basis of blood group typing variation has become better understood. As a result, nearly all clinical applications of blood group typing could be converted from serology to DNA testing (3).

SUMMARY

An embodiment of the disclosure is a microarray chip for performing blood group typing at a DNA level comprising a substrate; probes bound to the substrate; and a raw sample comprising DNA. In an embodiment, the raw sample is an air-dried cheek swab. In an embodiment, the raw sample is blood. In an embodiment, there are ABO-Rh probes. In an embodiment, the probe combination is such that a Rh-reaction will only occur if a Rh-deletion is present. In an embodiment, there are Weak D probes. In an embodiment, the probes are selected from SEQ ID NO: 163-180. In an embodiment, there are Minor Antigen probes. In an embodiment, the probes are selected from SEQ ID NO: 45-68 and SEQ ID NO: 105-126.

An embodiment of the disclosure is a method of performing blood group typing comprising obtaining a raw sample from an individual; amplifying a target sequence to obtain an amplified target sequence; labeling the amplified target sequence to obtain a labeled amplified target sequence; adding the labeled amplified target sequence to a microarray chip; hybridizing the labeled amplified target sequence to at least one probe present on the microarray chip; washing the microarray chip; and measuring fluorescence of the microarray chip. In an embodiment, the raw sample is an air-dried cheek swab. In an embodiment, the method further comprises preparing the air-dried cheek swab from the individual by a rapid 30 min soak in an aqueous release buffer; wherein amplification of blood group loci occurs by PCR from the soaking product; and wherein the labeling is with a fluorophore by PCR to generate single-stranded DNA. In an embodiment, the raw sample is blood.

An embodiment of the disclosure is a computer program for performing complex blood group typing at the DNA level utilizing the method; wherein the software is installed on a computer. In an embodiment, the computer is part of a scientific instrument. In an embodiment, the computer interacts with a scientific instrument.

An embodiment of the disclosure is a method of building a database of pre-qualified blood donors comprising providing registration information of an individual; providing a raw sample of the individual to a collection location; performing blood group typing on the raw sample; and adding the blood group typing to a database comprising the registration information of the individual. In an embodiment, the raw sample is a cheek swab sample. In an embodiment, the collection location is a laboratory. In an embodiment, the raw sample is mailed to the laboratory. In an embodiment, the database is searched for a desired blood group typing.

The foregoing has outlined rather broadly the features of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter, which form the subject of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the manner in which the above-recited and other enhancements and objects of the disclosure are obtained, a more particular description of the disclosure briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. Understanding that these drawings depict only typical embodiments of the disclosure and are therefore not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through the use of the accompanying drawings in which:

FIG. 1 depicts a comparison of a T-chip microarray and other DNA-based tests.

FIG. 2 depicts a process for coupling Raw Sample Genotyping (RSG) to low-cost T-chip microarray testing.

FIG. 3 depicts an ABO-Rh sub-assembly. FIG. 3A shows microarray hybridization probe locations and FIG. 3B shows primer design.

FIG. 4 depicts a typical T-chip microarray including T-chip microarray image data and automatic T-chip allelotype analysis.

DETAILED DESCRIPTION

The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present disclosure only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the disclosure. In this regard, no attempt is made to show structural details of the disclosure in more detail than is necessary for the fundamental understanding of the disclosure, the description taken with the drawings making apparent to those skilled in the art how the several forms of the disclosure may be embodied in practice.

The following definitions and explanations are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary 3rd Edition.

The inventors have demonstrated that by coupling two technologies, “Raw Sample Genotyping” and “Low Cost Microarray Manufacture”, it is possible to perform complex blood group typing at the DNA level, on air-dried cheek swabs (or finger prick blood) as a microarray test. The test has been named the “T-Chip” and bypasses the need for DNA extraction prior to analysis of the blood group type. A focus is to deliver that ability to obtain complex blood group typing as a new type of molecular epidemiology. By analogy to well-known studies such as the National Marrow Donor Program (NMDP), a goal of this disclosure is to enable a very large donor population to be pre-screened at home, with a cheek swab, so that those donors would then stand ready to donate blood, much as NMDP volunteers are screened with a cheek swab to obtain their HLA-type for marrow donation (1).

A relatively small number of venous blood samples and cheek swabs from volunteers with a known blood type were tested. Only the principal blood types of clinical significance (ABO, Rh) were known for these samples and thus the highest level of technical refinement has been obtained for those more standard blood types. Additional work was completed for a number of minor blood types of secondary import, using synthetic gene (SG) fragments. The (SG) data indicate that the minor blood types can be analyzed directly from raw blood or raw cheek swabs in a way that bypasses DNA extraction.

It is now possible to obtain high resolution DNA-based blood typing in a clinic or blood bank via core-lab based sequencing and via hybridization-based analysis: using multiple qPCR tests (3), multiplexed solid state microarrays (5,6), or fluid-phase Luminex bead arrays (7). The potential value of both multiplexed microarray or Luminex testing has become highly attractive. Both platforms have been commercialized and are presently used by AABB certified blood banks and in some cases as the basis for clinical practice.

Two industry leaders are the Grifols ID CORExt test (Luminex based) and Immucor's Precise Type HEA (Bead-Chip microarray). These are compared to the T-Chip in FIG. 1.

The antigen coverage of all 4 tests is not the same. The T-chip test is more complete, measuring ABO, RHD and Weak D along with the minor antigens which are the primary focus of the other 3 DNA tests.

There are at least two differences between the T-Chip and the other three technologies. The T-Chip test supports a medical testing market that the other technologies cannot: namely, the deployment of DNA based blood group typing as the basis for Public Health Screening and Research Epidemiology of the Blood Group Type as a Disease Risk factor (8-10).

The first difference is Raw Sample Genotyping (RSG) enables low-cost field collection for blood group typing. RSG allows complex microarray testing to be performed on raw samples in the complete absence of DNA extraction and DNA characterization (11,12). Based on RSG, the T-Chip will be able to use cheek swabs (or a dried blood spot) as input for high throughput blood group typing. In an embodiment, using an inexpensive heat block, 100 swabs can be prepared simultaneously for T-Chip testing via a rapid 30-minute soak in an aqueous release buffer. In an embodiment, the soaking product is used, as-is, for PCR amplification of blood group loci, then labeled with a fluorophore (also by PCR) to generate single-stranded DNA that can be pipetted as-is from PCR tube and transferred without manipulation directly to the microarray (FIG. 2). The other tests (ID CORExt, Precise Type, HiFi) require DNA purification and characterization in their test workflow: which nearly doubles the labor, cost and time required to prepare samples for analysis, while also giving rise to significant DNA loss and dilution. Because of the DNA loss and dilution attendant to DNA purification, neither ID CORExt or Precise Type HEA or HiFi are qualified for swab-based collection, whereas the T-Chip test is optimized to exploit the use of such raw swabs. The ability to use ordinary cheek swabs, with little-to-no sample preparation will position the T-Chip test as a unique technology solution for swab based (epidemiological) field applications of DNA-based blood group typing.

The second difference is microarray technology enables low-cost microarray analysis for blood group typing. The microarray technology allows DNA microarrays of the complexity required for blood group typing (@350 probes) to be mass produced at a rate of several thousand arrays per day, at a cost per microarray that is roughly ¼th the price per test of the Luminex based (ID CORExt), or Bead Array (Precise type), or Plate Based Array (HiFi) test: thereby dropping test consumable cost by a factor of @5. In addition, the microarray technology allows DNA hybridization (which is the basis for all 4 tests in FIG. 1) to be performed at lab ambient temperature without the need for temperature control or fluidics other than a simple pipette tip. Via that simplification, the T-Chip test is performed without any specialized lab equipment: whereas the Grifols Test requires Luminex fluidics (@$100K) and the Immucor and AXO test each require a highly-specialized microfluidics delivery system (also @$100K). The only specialized equipment required for the T-Chip is a generic fluorescence scanner from Sensovation for a cost of <$20K). The resulting drop in reagent cost by @5-fold, while also dropping the cost of ancillary equipment by more than a factor of 3 is also an enabling aspect of the T-Chip technology. FIG. 2.

The T-Chip test could enable fundamentally new aspects to the clinical and research utility of blood banks. Namely, the ability to pre-screen a very large donor community via a combination of web-site registration, cheek swab sample collection, and mail-in sample transport to an AABB (formerly known as the American Association of Blood Banks) laboratory: where the T-Chip technology would allow hundreds of samples a day to be collected and processed to generate complex DNA blood group profiles, which could grow to become a large regional database of pre-qualified donors.

In an embodiment, a proposed model is for a very low cost, community-scale blood group database-building. Once developed and deployed, the goal for the T-Chip test is to support targeted blood unit delivery for clinical practice and to support research into the role of blood group marker variation as a biomarker for disease risk.

One technology utilized here is Raw Sample Genotyping “RSG” technology, comprising the “front-end” of the T-Chip test. Another technology teaches the “back-end” of the T-Chip test, namely mass-production of DNA microarrays which are not only low-cost but display sensitivity and specificity near the theoretical limit defined by nucleic acid biophysics.

Three different products which perform DNA-based blood group typing are already on the market, based on Luminex beads and two different kinds of microarray technology (5-7). They were developed to analyze purified DNA from a venous blood draw, and therefore were well-positioned to be a routine test in an AABB blood bank.

The T-Chip test will also work with purified DNA and would be a simpler, much less expensive, and more accurate option than the three products above.

In an embodiment, a focus is to create a completely new population-scale market for DNA-based blood group typing wherein blood group typing can be based on inexpensive swab-based sample collection, followed by the elimination of all DNA purification steps, and analysis on a microarray platform which is inexpensive enough to support DNA based typing at a cost that is about the same as DNA-based microbial testing. In an embodiment, the initial deployment of the T-Chip will be to enable very large-scale pre-qualification of potential blood donors. In an embodiment, the T-Chip could evolve to be used to support universal blood group typing at birth (on the same Guthrie cards used since 1962) or as the basis for national-scale blood group typing in resource-limited markets such as Africa and South America. Realistically, not one of the current sets of predicate tests could address those important public-health-scale markets because they are too expensive in terms of labor and consumables.

T-Chip Design Principles. In an embodiment, the T-Chip microarray will accommodate the DNA from a single unpurified cheek swab as sample input, under conditions where the resulting steps in the microarray test (e.g., hybridization, washing, and data analysis) can be executed at room temperature by any lab technician, without special expertise or equipment other than an inexpensive optical scanner.

In an embodiment, a version of the “watchmaker's” decomposition into “sub-assemblies” was utilized. In an embodiment, the full set of blood typing tests was resolved into 4 multiplex PCR reactions with cognate microarray probe design to go with each. In an embodiment, each PCR reaction converts 2 μL of a raw swab eluate into a sample that is ready for microarray testing. Thus, a single swab (which yields @30 μL of eluate) can support at least 3 repeats of the entire T-Chip test. Target gene sub-assembly decomposition is as follows (I) ABO-Rh, (II) Minor Allele Variants, Group #1, (III) Minor Allele Variants, Group #2 and Weak D: (i.e., those genetic changes generating a subtle change in the Rh+ serotype not related to overt Rh deletion).

ABO-Rh. Another focus is on the ABO-Rh sub-assembly. This focus is for at least three reasons: 1. The ABO-Rh grouping defines much of blood group typing as ordinarily deployed. 2. Although ABO analysis is a relatively simple SNP design problem, the Rh+/− genotype is an unusually complex analytical problem, in that most Rh-serotypes are derived from a @1 kb long block deletion within the Rh gene. A key requirement for such Rh+/− discrimination, especially in a heterozygote, is to convert the 1 kb block deletion into a positive microarray signal, rather than a simple loss of copy number. 3. The ABO-Rh problem is sufficiently difficult that the predicate tests did not include it, choosing to focus on the minor alleles only.

To convert the Rh-block deletion into a positive microarray signal, a PCR-microarray probe combination has been designed such that (Rh-) PCR reaction will only occur if the Rh-deletion is present. Consequently, the Rh-deletion creates two redundant microarray probe signals which only occur upon deletion: the result being that the Rh+/− heterozygote (obtained by the standard Rh-deletion) can now be unambiguously resolved, along with full ABO typing. FIG. 3. ABO-Rh Sub-Assembly: Primer Design & Microarray Hybridization Probe Locations.

The Minor Allele Variants #1, #2: In Tables I and II, the Minor Allele variants have been grouped into two sub-assemblies for the purpose of PCR amplification. For the first time, T-Chip microarray data for both sets (Tables III and IV), using custom made gene-sized DNA fragments (made by Synthetic Genetics Technology) are shown, which each present the known clinically relevant SNP changes (13).

TABLE I
Minor Blood Group #1, #2: Primer Design & Microarray Probe Locations. Probe and Primer Sequences
for Minor Allele Set #1
				PCR
				Product		Probe Specificity
		PCR Primer	Primer sequence	size	ASO Probe sequence	(Analyte or Allele name)
Minor Antigen	Primary PCR	RHCE Exon 2 1′FP	TGTGGCCTTCAACCTCTTCATGCTG	144 bp	AGTTCCCTCCTGG	307C (RHCE*c)
Multiplex PCR	Primers	RHCE Exon 2 1′RP	(Seq Tag)AATACCTGAACAGTGTGATGACCAC
Reaction 1	Secondary	RHCE Exon 2 2′FP	TCTTCATGCTGGCGCTTGGTGTGCA	130 bp	AGTTCCCTTCTGG	307T (RHCE*C, RHD)
	PCR Primers	Universal Tag Primer CY3	TTTTGACTAGGAAACAGCTATGACCATG
	Primary PCR	RHCE Exon 5 1′FP	TCTTGTGGATGTTCTGGCCAAGTGT	155 bp	TCAACTCTGCTCTG	676G (RHCE*e)
	Primers	RHCE Exon 5 1′RP	(Seq Tag)CCCTGAGATGGCTGTCACCACACTG		TCAACTCTCCTCTG	676C (RHCE*E)
	Secondary	RHCE Exon 5 2′FP	TTGTGGATGTTCTGGCCAAGTGTCA	153 bp	TATGCTCTAGCAGT	733C
	PCR Primers	Universal Tag Primer CY3	TTTTGACTAGGAAACAGCTATGACCATG		TATGCTGTAGCAGT	733G (VS)
	Primary PCR	Exon 7 1′FF	(Seq Tag)CTCCGTCATGCACTCCATCTTCAGC	139 bp	GCAGACCCAGCA (AS)	1006G
	Primers	RHCE Exon 7 1′RP	GACCCACATGCCATTGCCGTTCCAG
	Secondary	Universal Tag Primer CY3	TTTTGACTAGGAAACAGCTATGACCATG	130 bp	GCAGACACAGCA (AS)	1006T (V)
	PCR Primers	RHCE Exon 7 2′RP	GCCATTGCCGTTCCAGACAGTATGA
	Primary PCR	KEL Exon 6 1′FP	CTTGGAGGCTGGCGCATCTCTGGTA	120 bp	AACCGAACGCTGA	578c (k)
	Primers	KEL Exon 6 1′RP	(Seq Tag)GAAATGGCCATACTGACTCATCAGA
	Secondary	KEL Exon 6 2′FP	GAGGCTGGCGCATCTCTGGTAAATG	116 bp	TAACCGAATGCTGA	578T (K)
	PCR Prmmers	Universal Tag Prrmer CY3	TITTGACTAGGAAACAGCTATGACCATG
	Primary PCR	KEL Exon 8 1′FP	AGACCCAAGCAAGGTGCAAGAACAC	133 bp	CACTTCACGGCT	841C (Kpb)
	Primers	KEL Exon 8 1′RP	(Seq Tag)TGCCCTGTGCCCGCCGCTGCTCCAG
	Secondary	KEL Exon 8 2′FP	AGGTGCAAGAACACTCTTCCTTGTC	122 bp	CACTTCATGGCTG	841T (Kpa)
	PCR Primers	Universal Tag Primer CY3	TTTTGACTAGGAAACAGCTATGACCATG
	Primary PCR	KEL Exon 17 1′FP	CCCTATGTTCTCTTGCTGTATGTTC	141 bp	CTGCCTCGCCT	1790T (Jsb)
	Primers	KEL Exon 17 1′RP	(Seq Tag)TTCAGGCACAGGTGAGCTTCCTGGA
	Secondary	KEL Exon 17 2′FP	TTCTCTTGCTGTATGTTCTCTTGTC	134 bp	CTGCCCCGCCT	1790C (Jsa)
	PCR Primers	Universal Tag Primer CY3	TTTTGACTAGGAAACAGCTATGACCATG
	Primary PCR	Duffy Exon 2a 1′FP	(Seq Tag)GTGTGAATGATTCCTTCCCAGATG	121 bp	TGGCATCATAGTCT (As)	125A (Fyb)
	Primers	Duffy Exon 2a 1′RP	CAGAGTCATCCAGCAGGTTACAGGA
	Secondary	Universa1 Tag Primer CY3	TTTTGACTAGGAAACAGCTATGACCATG	114 bp	TGGCACCATAGTC (AS)	125G (Fya)
	PCR Primers	Duffy Exon 2a 2′RP	ATCCAGCAGGTTACAGGAGTGGCAG
	Primary PCR	Duffy Promotor 1′FP	CAGAACCTGATGGCCCTCATTAGTC	97 bp	GCTCTTATCTTGGA	−67 (Fyb)
	Primers	Duffy Promoter 1′RP	(Seq Tag)GGACGGCTGTCAGCGCCTGTGCT
	Secondary	Duffy Promotor 2′FP	ACCTGATGGCCCTCATTAGTCCTTG	93 bp	GCTCTTACCTTGG	−67 (FY*01N.01)
	PCR Primers	Universal Tag Primer CY3	TTTTGACTAGGAAACAGCTATGACCATG
	Primary PCR	Duffy Exon 2b 1′FP	GCTAGCAGCACTGTCCTCTTCATGC	105 bp	CTCTTCCGCTGG	265C (Fyb)
	Primers	Duffy Exon 2b 1′RP	(Seq Tag)AGGACAGGCCAGCCAGGGCAGAGCT
	Secondary	Duffy Exon 2b 2′FP	CACTGTCCTCTTCATGCTTTTCAGA	97 bp	TCTCTTCTGCTGG	265T FY*02M.01
	PCR Primers	Universal Tag Primer CY3	TTTTGACTAGGAAACAGCTATGACCATG
	Primary PCR	Kidd Exon 9 1′FP	(Seq Tag)TCTTAACAGGACTCAGTCTTTCAGC	138 bp	TAGATGTCCTCAAAT (AS)	838G (Jka)
	Primers	Kidd Exon 9 1′RP	AGAGAGCTGTTGAAACCCCAGAGTC
	Secondary	Universa1 Tag Primer CY3	TTTTGACTAGGAAACAGCTATGACCATG	132 bp	TAGATGTTCTCAAAT (AS)	838A (JKb)
	PCR Primers	Kidd Exon 9 2′RP	CTGTTGAAACCCCAGAGTCCAAAGT
	Primary PCR	MNS GYPA Exon 2 1′FP	TTCTCAACTTCTATTTTATACAGCA	183 bp	TCAGCATCAAGTAC	59C (M)
	Primers	MNS GYPA Exon 2 2′RP	(Seq Tag)AGATGTAACTCTTTGTGACTGAAGA
	Secondary	MNS GYPA Exon 2 2′FP	CTTCTATTTTATACAGCAATTGTGA	119 bp	TCAGCATTAAGTAC	59T (N)
	PCR Primers	Universal Tag Primer CY3	TTTTGACTAGGAAACAGCTATGACCATG
			SEQ ID NO: 1-44		SEQ ID NO: 45-68

TABLE II
Minor Blood Group #1, #2: Primer Design & Microarray Probe Locations.
Probe and Primer Sequences for Minor Allele Set #2
				PCR
				Product		Probe Specificity
		PCR Primer	Primer sequence	size	ASO Probe sequence	(Analyte or Allele name)
Minor Antigen	Primary PCR	MNS GYPB Exon 4 1′FP	AATGATTTTTTTCTTTGCACATGTC	143 bp	GAGAAACGGGACA	143C (s)
Multiplex PCR	Primers	MNS GYPB Exon 4 1′RP	(Seq Tag)AATATTAACATACCTGGTACAGTGA
Reaction 2	Secondary	MNS GYPB Exon 4 2′FP	TCTTTCTTATTTGGACTTACATTGA	120 bp	GAGAAATGGGACAA	143T (s)
	PCR Primers	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG
		Primer CY3
	Primary PCR	MNS GYPB Exon 5 1′FP	(Seq Tag)TTATTTTGTGTGTGATGGCTGGTAT	178 bp	GGATCGTTCCAATA (AS)	230C (GYPB)
	Primers	MNS GYPB Intron 5 1′RP	AACTCAGAGGAATAAACCCTCCTAG		GGATCATTCCAATAA (AS)	230T (GYP*NY, GYP*He(NY))
	Secondary	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG	153 bp	CTGAATTCTCACCT (AS)	Intron5 G (GYPB)
		Primer CY3
	PCR Primers	MNS GYPB Intron 5 2′RP	AGCTGTTCACACTGGTATTTAGAGC		CTGAATTATCACCTT (AS)	Intron5 T(GYP*NY, *He
						(NY),*P2, *He(P2)
	Primary PCR	Lu Exon 3 1′FP	GGACACCCGGAGCTGAGAGCCTGCC	123 bp	CCCCGCCTAGC	230G (LUb)
	Primers	Lu Exon 3 1′RP	(Seq Tag)GCTCAGAGCCCTGCATCTCAGCCGA
	Secondary	Lu Exon 3 2′FP	CCCGCGCCCACAGACCGACCGCTCG	100 bp	CCCCCACCTAGC	230A (LUa)
	PCR Primers	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG
		Primer CY3
	Primary PCR	Di Exon 19 1′FP	GGCATCCAGATCATCTGCCTGGCAG	127 bp	CACGCCGGCCT	2561C (Dib)
	Primers	Di Exon 19 1′RP	(Seq Tag)GCAGCGGCACAGTGAGGATGAGGAC
	Secondary	Di Exon 19 2′FP	CAGATCATCTGCCTGGCAGTGCTGT	121 bp	CACGCTGGCCT	2561T (Dia)
	PCR Primers	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG
		Primer CY3
	Primary PCR	Co Exon 1 1′FP	(Seq Tag)CTGCCCTGGGCTTCAAATACCCGGT	119 bp	GGACCGCCGTC (AS)	134C (Coa)
	Primers	Co Exon 1 1′RP	GATGCTCAGCCCGAAGGCCAGCGAC
	Secondary	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG	105 bp	GGACCACCGTCT (AS)	134T (Cob)
		Primer CY3
	PCR Primers	Co Exon 1 2′RP	AGGCCAGCGACACCTTCACGTTGTC
	Primary PCR	Dom Exon 2 1′FP	(Seq Tag)GCTGTTTAAAGTTATAAATATGAGC	123 bp	ACCAGTTTCCTCT (AS)	793A (Doa)
	Primers	Dom Exon 2 1′RP	AGCTGACAGTTATATGTGCTCAGGT
	Secondary	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG	113 bp	ACCAGTCTCCTCT (AS)	793G (Dob)
		Primer CY3
	PCR Primers	Dom Exon 2 2′RP	TATATGTGCTCAGGTTCCCAGTTGA
	Primary PCR	Dom Exon 2 1′FP	AGAAGAATTATTTTAGGATGTGGCA	142 bp	ACCAAGGAAAAGTT	323G (Hy+)
	Primers	Dom Exon 2 1′RP	(Seq Tag)GTYTAAAACAAAATAGCCACAGCGT		ACCAAGTAAAAGTTC	323T (Hy−)
	Secondary	Dom Exon 2 2′FP	GGCAAAAAGCCCACTTAGCCTGGCT	123 bp	ATGACTACCACACA	350C Jo(a+)
	PCR Primers	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG		ATGACTATCACACA	350T Jo(a−)
		Primer CY3
	Primary PCR	Lw Exon 1 1′FP	(Seq Tag)CTGCGGCAAGGCAAGACGCTCAGAG	131 bp	AGCAGCTGGTAAG (AS)	299A (LWa)
	Primers	Lw Exon 1 1′RP	GCGCAGGTCACGAGGCAGTGCGCGA
	Secondary	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG	118 bp	GCAGCCGGTAAG (AS)	299G (LWb)
		Primer CY3
	PCR Primers	Lw Exon 1 2′RP	GGCAGTGCGCGAGGGAGCTCCAGGC
	Primary PCR	Sc Exon 4 1′FP	(Seq Tag)TTGGGCACAGCCGAGCTGCTCTGCC	150 bp	ACCGTCCCGGG (AS)	169G (Sc1)
	Primers	Sc Exon 4 1′RP	CATCCCGGAATATGTGAACAGCCTG
	Secondary	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG	133 bp	ACCGTCCTGGG (AS)	169A (Sc2)
		Primer CY3
	PCR Primers	Sc Exon 4 2′RP	ACAGCCTGGGAGCGCTGCGGGAATG
			SEQ ID NO: 69-104		SEQ ID NO: 105-126

TABLE III
T-Chip Data: Minor Allele Set #1
	Hybridization Signal from	Hybridization Signal from
	Perfect Match Allele	Alternate Allele Target	Ratio
Probe/specificity	Target(RFU)	(RFU)	PM:MM
RHCE*c	307C	65535	857	76:1
RHCE*C, RHD	307T	65407	4290	15:1
RHCE*e	676G	32860	6005	5:1
RHCE*E	676C	65535	696	94:1
RHCE	733C	1000	80	13:1
RHCE*VS	733G	856	95	9:1
RHCE	1006G	65535	447	147:1
RHCE*V	1006T	59052	9635	6:1
KEL k	578C	65535	1341	49:1
KEL K	578T	35283	19325	2:1
KEL Kpb	841C	65535	795	82:1
KEL Kpa	841T	19417	1369	14:1
KEL Jsb	1790T	65535	9360	7:1
KEL Jsa	1790C	65535	5418	12:1
Duffy Fyb	125A	65535	6814	10:1
Duffy Fya	125G	65535	4782	14:1
Duffy Fyb	−67T	65535	5171	13:1
Duffy (FY*01N.01)	−67C	65535	935	70:1
Duffy Fyb	265C	65535	4835	14:1
Duffy (FY*02M.01)	265T	46764	13947	3:1
Kidd Jka	838G	58770	1051	56:1
Kidd Jkb	838A	51771	2278	23:1
MNS M	59C	46418	381	122:1
MNS N	59T	30045	1455	21:1

TABLE IV
T-Chip Data: Minor Allele Set #2
	Hybridization Signal from	Hybridization Signal from
	Perfect Match Allele	Alternate Allele Target	Ratio PM
Probe/specificity	Target(RFU)	(RFU)	vs. MM
MNS s	143C	18275	294	62:1
MNS S	143T	11960	323	37:1
MNS	230C	18342	393	47:1
MNS NY, HeNY	230T	22564	622	36:1
MNS	IN5G	11779	193	61:1
(GYP*NY, *He(NY),	IN5T	46230	545	85:1
*P2, *He(P2)
Lub	230G	65535	18036	4:1
Lua	230A	20429	234	87:1
Dib	2561C	2606	116	22:1
Dia	2561T	44652	6633	7:1
Coa	134C	16709	875	19:1
Cob	134T	7040	416	17:1
Doa	793A	35017	921	38:1
Dob	793G	49339	444	111:1
Do (Hy+)	323G	20862	1053	20:1
Do (Hy−)	323T	29129	713	41:1
Jo (a+)	350C	54381	747	73:1
Jo (a−)	350T	30492	3118	10:1
Lwa	299A	522	129	40:1
Lwb	299G	5494	113	49:1
Sc1	169G	1688	206	8:1
Sc2	169A	59902	4347	14:1

The data shown Tables III and IV are T-Chip hybridization data for both the Minor Allele #1 (Table III) and Minor Allele #2 (Table IV) sub-assemblies. Generally, the specificity is very high (match/single mismatch >10). However a small number of probes [Duffy FY*02M01, Lub, Dia, Sc1] show lower specificity in the 4-10 range, which is not acceptable. Work is in progress to increase the performance of that small number via probe shortening.

Weak D

Substantial effort has begun to suggest that the so called “Weak D” serology (i.e., serological phenotypes in-between Rh+& Rh-) should be complemented by genetic analysis to aid in early treatment of the neonate (14-17).

A new “Weak D” sub-assembly into the design of the T-Chip microarray is included based on analysis of the following set of markers: Weak D types 1, 2 and 3. All markers can be resolved via simple SNP analysis, at a level of complexity that is a bit simpler than Minor Antigen Sets #1 or #2 (Tables V and VI). As is the case for ABO-Rh, none of the 3 commercialized Predicate Tests can generate Weak D data (FIG. 1).

TABLE V
Weak D BLood Group: Primer Design & Microarray Probe Locations
				PCR
				Product		Probe Specificity
		PCR Primer	Primer sequence	size	ASO Probe sequence	(Analyte or Allele name)
Weak D and Partial D	Primary PCR	RH* Exon 1 1′FP	TGCCTGGTGCTGGTGGAACCCCTGC	83 bp	TGAGCTCTAAGTAC	8C (RHD, RHCE)
Multiplex PCR	Primers	RH* Exon 1 1′RP	(Seq Tag)GGCAGGCAGCGCCGGACAGACCGC
Reaction	Secondary	RH* 5′NCR 2′FP	TGGTGCTGGTGGAACCCCTGCACAG	79 bp	TGAGCTGTAAGTAC	8G (Weak D Type 3)
	PCR Primers	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG
		Primer CY3
	Primary PCR	RHD Exon 2 1′FP	CGAGCAGTTGGCCAAGATCTGACCG	81 bp	TGGCTTGGGCTT	186G (RHD)
	Primers	RHD Exon 2 1′RP	(Seq Tag)CCAGCTGTGTCTCCGGAAACTCGAG
	Secondary	RHD Exon 2 2′FP	AGTTGGCCAAGATCTGACCGTGATG	76 bp	TGGCTTTGGCTTC	186T (DIIIa, DIVa)
	PCR Primers	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG
		Primer CY3
	Primary PCR	RHD Exon 3 1′FP	(Seq Tag)TGCTTTGTCGGTGCTGATCTCAGTG	119 bp	AACTGCGCCAAGT(AS)	410C (RHD)
	Primers	RHD Exon 3 1′RP	TTACTGATGACCATCCTCAGGTTGC
	Secondary	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG	109 bp	AACTGCACCAAGTT(AS)	410T (DIIIa, DIVa)
		Primer CY3
	PCR Primers	RHD Exon 3 2′RP	CATCCTCAGGTTGCCTAAAGCTGTC
	Primary PCR	RHD Exon 4 1′FP	(Seq Tag)CTACCCGAGGGAACGGAGGATAAAG	122 bp	GTTGCTGTCTGAT (AS)	602C ((+)RHD, DIVa),
	Primers	RH* Exon 4 lRP	AGCCATTCTGCTCAGCCCAAGTAGG			((−)DVI)
	Secondary	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG	81 bp	GTTGCTCTCTGAT (AS)	602G (DIIIa, DAR)
		Primer CY3
	PCR Primers	RHD Exon 4 2′RP	CCCCACCTTGTCCTTACCCAGCATG
	Primary PCR	RHD Exon 5 1′FP	CTTGTGGATGTTCTGGCCAAGTTTC	91 bp	TCCAATCGAAAGGA	697G ((+)RHD),((−)DVI)
	Primers	RH* Exon 5 1′RP	(Seq Tag)TACAGCATAGTAGGTGTTGAACACG
	Secondary	RHD Exon 5 2′FP	TGTTCTGGCCAAGTTTCAACTCTGC	83 bp	CCAATCCAAAGGAA	697C DV type1
	PCR Primers	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG		CCAATCAAAAGGAA	697A DV type5
		Primer CY3
	Primary PCR	RH* Exon 6 1′FP	(Seq Tag)TTACCCACACGCTATTTCTTTGCAG	179 bp	CTGTGCACATAAGT (AS)	809T (RHD)
	Primers	RHD Exon 6 1′RP	TGTCTAGTTTCTTACCGGCAGGTAC
	Secondary	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG	165 bp	CTGTGCCCATAAG (AS)	809G (Weak D Type1)
		Primer CY3
	PCR Primers	RHD Exon 6 2′RP	CGGCAGGTACTTGGCTCCCCCGACG
	Primary PCR	RHD Exon 7 1′FP	ATTCCCCACAGCTCCATCATGGGCT	113 bp	TGCTTGATACCGT	1048G (RHD)
	Primers	RHD Exon 7 1′RP	(Seq Tag)CCCACATGCCATTGCCGGCTCCGAC
	Secondary	RHD Exon 7 2′FP	CTCCATCATGGGCTACAACTTCAGC	102 bp	GTGCTTCATACCG	1048C (DIVa)
	PCR Primers	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG
		Primer CY3
	Primary PCR	RH* Exon 8 1′FP	GGATTGGCTTCCAGGTCCTCCTCAG	85 bp	CCATCGTGATAGCTCTC	(+)1136C (RHD)
	Primers	RHD Ex8 1136C 1′RP	(Seq Tag)CTGACCTGTCAGGAGACCAGACATG
	Secondary	RH* Exon 8 2′FP	GGCTTCCAGGTCCTCCTCAGCATTG	80 bp		(−)(RHD 1136C absent (DAU)
	PCR Primers	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG
		Primer CY3
	Primary PCR	RHD Exon 9 1′RP	AAGGATTTCTGTTGAGATACTGTCG	151 bp	AAACAGGTTTGCTC	1154G (RHD)
	Primers	RHD Exon 9 1′RP	(Seq Tag)CATGAGGTGCTTTCCATATTTTAAG
	Secondary	RHD Exon 9 2′FP	TGTCGTTTTGACACACAATATTTCG	131 bp	AAACAGCTTTGCTC	1154C (Weak D type 2)
	PCR Primers	Universal Tag	TTTTGACTAGGAAACAGCTATGACCATG
		Primer CY3
			SEQ ID NO: 127-162		SEQ ID NO: 163-180

TABLE VI
T-Chip Microarray Manufacture and Consumable Kits
	Discrete	Total Probes
	DNA Probes	(Triplicate)
Gene Loci	in T-Chip	in T-Chip
ABO, Rh	20	60
Weak D	24	72
Minor Antigens #1, #2	50	150
Controls	14	42
Total	108	324
Number of	4	[ABO-Rh],
PCR reactions		[Minor Antigens #1],
Per T-Chip Test		[Minor Antigens #2],
		[Weak D]
Array Manufacture	Q-Array 2 (Tucson, AZ)	3240
(In House)	90 × 12 T-Chips	T-Chip Arrays/Week
	arrays per batch
	Up to 3 batches per week
Array Manufacture	MI (Huntsville AL)	18,000
(OEM)	500 × 12 T-Chip	T-Chip Arrays/Week
	Arrays per batch
	Up to 3 batches per week
PCR Kit manufacture	48 arrays per kit	100 kits/week
(In House)	Including all wsrt-ware	4800 array kits/week

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

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Claims

1. A microarray chip for performing blood group typing at a DNA level comprising a substrate;probes bound to the substrate; anda raw sample comprising DNA.

2. The microarray chip of claim 1 wherein the raw sample is an air-dried cheek swab.

3. The microarray chip of claim 1 wherein the raw sample is blood.

4. The microarray chip of claim 1 wherein there are ABO-Rh probes.

5. The microarray chip of claim 4 wherein the probe combination is such that a Rh-reaction will only occur if a Rh-deletion is present.

6. The microarray chip of claim 1 wherein there are Weak D probes.

7. The microarray chip of claim 6 wherein the probes are selected from SEQ ID NO: 163-180.

8. The microarray chip of claim 1 where there are Minor Antigen probes.

9. The microarray chip of claim 8 wherein the probes are selected from SEQ ID NO: 45-68 and SEQ ID NO: 105-126.

10. A method of performing blood group typing comprising obtaining a raw sample from an individual; amplifying a target sequence to obtain an amplified target sequence;labeling the amplified target sequence to obtain a labeled amplified target sequence;adding the labeled amplified target sequence to a microarray chip;hybridizing the labeled amplified target sequence to at least one probe present on the microarray chip;washing the microarray chip; andmeasuring fluorescence of the microarray chip.

11. The method of claim 10 further comprising preparing an air-dried cheek swab from the individual by a rapid 30 min soak in an aqueous release buffer; wherein amplification of blood group loci occurs by PCR from the soaking product; andwherein the labeling is with a fluorophore by PCR to generate single-stranded DNA.

12. The method of claim 10 wherein the raw sample is blood.

13. A computer program for performing complex blood group typing at the DNA level utilizing the method of claim 10; wherein the software is installed on a computer.

14. The computer program of claim 13, wherein the computer is part of a scientific instrument.

15. The computer program of claim 13, wherein the computer interacts with a scientific instrument.

16. A method of building a database of pre-qualified blood donors comprising providing registration information of an individual;providing a raw sample of the individual to a collection location;performing blood group typing on the raw sample; andadding the blood group typing to a database comprising the registration information of the individual.

17. The method of claim 16 wherein the raw sample is a cheek swab sample.

18. The method of claim 16 wherein the collection location is a laboratory.

19. The method of claim 18 wherein the raw sample is mailed to the laboratory.

20. The method of claim 16 wherein the database is searched for a desired blood group typing.