US 20090186340A1
(19) United States (12) Patent Application Publication (10) Pub. N0.: US 2009/0186340 A1 Olsson et al. (54)
(43) Pub. Date:
GENE ANALYSIS
(75) Inventors:
(86)
Martin Lennarth Olsson, Lund (SE); Jill Rosalind Storry, Lund
PCT No.:
PCT/GB05/03659
§ 371 (6X1), (2), (4) Date/1
(SE); Neil David Avent, Bristol
(GB); Tracey Elizabeth Madgett, Bristol (GB) BANNER & WITCOFF, LTD.
App1,No,;
(22) PCT Filed;
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_
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Pubhcatm“ Classl?catm“
(51) Int. Cl.
UNIVERSITY OF THE WEST OF ENGLAND, BRISTOL, Bristol (GB); UNIVERSITETSS 1
(21)
_
(GB) ................................. .. 0421136.3 (GB) ................................. .. 05059837 _
1100 13th STREET, N.W., SUITE 1200
(73) Assignees:
_
Forelgn Appllcatwn Prwrlty Data
Sep. 22, 2004 Mar. 23, 2005
Correspondence Address.
WASHINGTON, DC 20005-4051 (US)
Feb- 2, 2009 _
(30)
Jul. 23, 2009
52
C12Q 1/68
(2006.01)
C07H 21/04 C40B 40/08
(2006.01) (2006.01)
U s C]
LUND, BLODCENTRALEN
(
)
SKANE, Lund (SE)
(57)
.
.
435/6' 536/24 33- 506/17 . ........................... ..
,
.
,
ABSTRACT
11/663,624
This invention relates to a series of PCR primers that Will alloW the simultaneous ampli?cation of regions of the clini
Sq), 22, 2005
cally signi?cant ABO and RHD genes.
Patent Application Publication
Jul. 23, 2009 Sheet 1 0f 26
101F/
intlF
303F/
403F/
502F/
60lF/ 702F/
198R
/297R
397R
499R
599R
697R
01F
US 2009/0186340 A1
901F
799R
Key: Shaded boxes = eXons; numbers refer to exon number
Each box has forward primer/reverse primer detailed Reverse primers for exons 2, 4 and 5 span at least some of the 3’ end of the exon in
question; all other primers are in the intron sequence
RHD Exon 5 will only be ampli?ed from RHD RQHar genomic DNA samples by using an RHCE-speci?c intron 4 forward primer pairing With the RHD-speci?c reverse
primer (599R)
Patent Application Publication
Jul. 23, 2009 Sheet 2 0f 26
US 2009/0186340 A1
Fig. 2A Exon 1 RHCE : RHD :
CTTCCGTGTTAACTCCATAGACAGGCCAGCACAGCCAGCC'I‘TGCAGCCTGAGATAAGGCCTTTGG CTTCCGTGTTAACTCCATAGAGAGGCCAGCACAACCAGCCTTGCAGCCTGAGATAAGGCCTTTGG
CTTCCGTGTTAACTCCATAGA AGGCCAGCACA CCAGCCTTGCAGCCTGAGATAAGGCCTTTGG RHCE : RHD :
CGGGTGTCTCCCCTATCGCTCCCTCAAGCCCTCAAGTAGGTGTTGGAGAGAGGGGTGA'I‘GCCTGG CGGGTGTCTCCCCTATCGCTCCCTCAAGCCCTCAAGTAGGTGTTGGAGAGAGGGGTGATGCCTGG
CGGGTGTCTCCCCTATCGCTCCCTCAAGCCCTCAAGTAGGTGTTGGAGAGAGGGGTGATGCCTGG RHCE :
RHD
TGCTGGTGGAACCCCTGCACAGAGACGGACACAGGATGAG'CTCTAAGTACCCGC'GGTCTGTCCGG
: TGCTGGTGGAACCCCTGCACAGAGACGGACACAGGATG'AGCTQTAAGTACCCGCGGTCTGTC'CG'G
TGCTGG'I'GGAACCCCTGCACAGAGACGGACACAGGATGAGCTCTAAGTACCC'GCGGTC'TGTCCGG RHCE : CGCTGCCTGCCCCTCTGQGCCCTAACACTGGAAGCAGCTCTCATTCTCCTCTTCTATTTTTTTAC RHD : CGCTGCC'TGC'CCCTCTGQGCCCTAACACTGGAAGC'AGCTCTCATTCTC'C'TCTTCTATTTTTTTAC CGCTGCCTGCCCCTCTG GCCCTAACACTGGAAGCAGCTCTC'ATTCTCCTCTTCTATTTTTTTAC RHCE :
CCACTATGACGCTTCCTTAGAGGATCAAAAG'GGGCTCGTGG'CQATCCTATCAAGGTGAGAGTTCAT
RHD
C'CAC'TATGACGCTTCC‘TTAGAGGATgAAAAGGGGCTCGTGGCATCCTATC'AAGGTGAGAGTTCAT
:
CCACTATGACGCTTCCTTAGAGGATCAAAAGGGGCTCGTGGCATCCTATCAAGGTGAGAGTTCAT RHCE :
TGGAACAGTGGTCACAGGAGCAAATAGCAGGGGCAGGGGCGGGGGAGGCCTATGGTTCTCCAGGG
RHD
TGGAAAAGTGGTCACAGGAGCAAATAGCAGGGGCAGGGGCGGGGGAGGCCTGTGGTTC'I‘CCAGGG
:
TGGAA AGTGGTCACAGGAGCAAATAGCAGGGGCAGGGGCGGGGGAGGCCT TGGTTCTCCAGGG RHC'E : RHD :
GCACAGATGTTCCTTTCTACAAAATCCCGAGGAAAA-GATTCCCCCATCTTCTTCCGTAGATTGC GCACAGATGTTCCTTTCTACAAAATCCCAAGGAAAAAGATTCCCCCATCT’I‘CTTCCGTAGATTGC
GCACAGATGTTCCTTTCTACAAAATCCC AGGAAAA GATTCCCCCATCTTCTTCCG'I‘AGATTGC
Key: exon : italics
primers : bold differences between RHD and RHCE in exon : underlined SNPs for variant RHD alleles : double underlined
Consensus sequence is shown underneath each set of RHCE/RHD sequence.
Patent Application Publication
Jul. 23, 2009 Sheet 4 0f 26
US 2009/0186340 A1
RHCE : GCTGCCATTTAG'I'AAGACTCTAA‘I‘TTCATACCACCCTAAATCTCGTCTGCTTCCCCCTCCTCCTT RHD : GCTGCCATTTAGTAAGACTCTAATTTCATACCACCCTAAATCTCGTCTGCTTCCCCCTCGTCCTT
GCTGCCATTTAGTAAGACTCTAATTTCATACCACCCTAAATCTCGTCTGCTTCCCCCTC TCCTT RHCE : CTCACCATCTCCCCACCGAGCAGTQGGCCAAGATCTGACCGTGATGGCGGCCQTTGGCTTGGGCT RHD : CTCGCCATCTCCCCACCG‘AGCAGglIGGCCAAGATCgGACCGTGATGGCGGCCéTTGGCTTg'GGCT CTC CCATCTCCCCACCGAGCAGT GGCCAAGA'I‘CTGACCGTGATGGCGGCC TTGGCTTGGGCT RHCE : TCCTCACCTCéAéTTTCCGGAGACACAGC'TGGAGCAGTG'TGGCCTTCAACCTC'TTCATGCTGGCG RHD : TCCTCACCTCQAQTTTCCGGAGACACAGCTGGAGCAGTGTGGCCTTCAACCTCTTCATGCTGGCG TCCTCACCTC A T'I'TCCGGAGACACAGCTGGAGCAGTGTGGCCTTCAACCTCTTCATGCTGGCG RHCE :
RHD
CTTGGTGTGCAGTGGGCAATCCTG'CTGGACGGCTTCC'TGAGCC'AGTTC'CCTECTGGGAAGGTGG'T
: CTTGGTGTGCAGTGEGCAATCCTGC'TGGACGGCTTCCTGAGCCAGTTCCCTZCTGGGAAGGTGGT
CT'I‘GGTGTGCAGTGGGCAATCCTGCTGGACGGC'I‘TCCTGAGCCAGTTCCCT CTGGGAAGGTGGT RHCE :
RHD
CATCACACTGTTCAGGTATTGGGATG'GTGGCTGGATCACTTCTGGGTCATAGAGGGAATGGACCC
: CATCACAClGTTCAigGTATTC-FGGATGGTGGCTGGATCAC'TTC‘I‘GGGTCATAGAGGGAATGGACCC
CATCACACTGTTCAGGTATTGGGATGGTGGCTGGATCACTTCTGGGTCATAGAGGGAATGGACCC
Key: exon : italics
primers : bold differences between RHD and RHCE (little c) in exon : underlined SN1’s for variant RHD alleles : do_ublel1_nde@1ll ?rst and last nucleotide of exon :
Consensus sequence is shown underneath each set of RHCE/RHD sequence. RH little 0 sequence is shown; RH big C sequence is highly homologous to RHD sequence in this region
Patent Application Publication
Jul. 23, 2009 Sheet 6 of 26
US 2009/0186340 A1
Fig 2D Exon 4
RHCE : TGGGTTGGGCTGGGTAAGCTCTGAACACC'AGTCTCGTGGCTTCAAGTCACACCTCCTAAGTGAAG RHD : TGGGTTGGGCTGGGTAAGCTCTGAACACCAGTCTCATGGCTTCAAGTCACACCTCCTAAGTGAAG
TGGGTTGGGCTGGGTAAGCTCTGAACACCAGTCTC TGGCTTCAAG'I'CACACCTCCTAAGTGAAG RHCE : CTCTGAACTTTCTCCAAGGACCATCAGGGCTTTCCCCTGGGCAGAGGATGCCGACACTCACTGCT RHD : CTCTGAACTTTCTCCAAGGACTATCAGGGCTTGCCCC—GGGCAGAGGATGCC'GACACTCACTGCT
CTCTGAACTTTCTCCAAGGAC ATCAGGGCTT CCCC GGGCAGAGGATGCCGACACTCACTGCT
RHCE 2 CTTACTGGGTTTTATTGCAG?CAG'AC'TACC'ACATGAACSTGAQGCACITCTAC'GTGTTCGCAGCC RHD
2 CTTAC'I‘GGGTTTTATTGCAGEZA‘CAGACTACCéCATGAAC'éTGAgGCAC'QTCTACG'TGTTCG'CAGCC CTTAC'I‘GGGTT'I‘TATTGCAGACAGACTACCACATGAAC TGA GCAC TCTACGTGTTCGCAGCC
RI-ICE : TATTTTGGGCTGéCTG'I'GGCCTGGTGC'CTGCCAAAGCCTCTACCCéAGC-IGAACGGAGGATAAIGA RHD : TATTTTGGGCTGICTGTGGCCTGGTGC'CTGCCAAAGCCTCTACCC'QAGGGAACGGAGGATAAéGJ-l
TA’I‘TTTGGGCTG CTGTGGCCTGGTGCCTGCCAAAGCCTCTACCC AGGGAACGGAGGATAA GA} RHCE : TCAGAQAGCAACGATAC'C'C'AGTTTGTCTGCCATGCTGGGTAAGGACAAGGTGGGG'TGAGTGGTCT
RHD
: TC'AGAQAGCAACGATAC'CCAGTTTGTC'TGC'CATGCTGGQTAAGGACAAGGTGGGGTGAGTGGTC'I‘ TCAGA AGCAACGATACCCAGTTTGTCTGCCATGCTGGGTAAGGACAAGGTGGGGTGAGTGGTCT
RHCE : CATACTTGGGCTGAGCAGAATGGCTCAGAAAAGGCTCTGGCTGAAAAAATCTCCCTCCTTTACCA RHD : CCTAC‘TTGGGCTGAGCAGAATGGCTCAGAAAAGGCTCTGGCTGAAAAAATCTCCC’I'CC'I'TTACCA
C TACTTGGGCTGAGCAGAATGGCTCAGAAAAGGCTCTGGCTGAAAAAATCTCCCTCCTTTACCA
Key: exon : italics
primers : bold differences between RHD and RHCE in exon : underlined SNPs for variant RHD alleles : double underlined ?rst and last nucleotide of exon are aid
Consensus sequence is shown underneath each set of RHCE/RHD sequence.
Patent Application Publication
Jul. 23, 2009 Sheet 8 of 26
US 2009/0186340 A1
rhce : AGCCATCTCAG'GG'TCATCCTTGGCTCACCCCCAAilGGAAGATCAGCAIGGTGAGCAGGGCGCTGC
rhd
: AGCCATCTCAGGGTCATCCTTGGCTCAC'C’C'CCAAgGGAAGATCAGCAggGTG-‘AGCAGGGCGCTGC AGCCATCTCAGGGTCATCCTTGGCTCACCCCCAA GGAAGATCAGCA GGTGAGCAGGGCGCTGC
rhce : rhd :
CCT'I'GGGCAGCAC'I'TGGGTCTAACAGGACTAGCACACATATTTATGCCCCTCCCCACCCCAGGGC CCTTGGGCAGCAC'I‘TGGGTCTAACAGGACTAGCACACATATTTATGCCCCTCCCCACCCCAGGGC
CCTTGGGCAGCACTTGGGTCTAACAGGACTAGCACACATATTTATGCCCCTCCCCACCCCAGGGC rhce : CAGCGTGGGTTGGGAGAGGACATGCCGGGTGGTGGAGCTGTGCCTGCCTCTACAGTGGAGCTCTA rhd : CAGCGTGGGT'I‘GGGAGAGGGCATGCCGGGTGGTGGAGCTGTGCCTGCCTCTACAGTGGAGCTCTA
CAGCGTGGGTTGGGAGAGG CATGCCGGGTGGTGGAGCTGTGCCTGCCTCTACAG'I‘GGAGCTCTA rhce : GGAAGAATGC'I'GGGTGGTCACAGGGGGCCTGGGACTCAGGAGACTGTCCAGTGA'I'CAAAGGCTTT rhd : GGTAGAATGC‘I‘GGG'I‘GG'I‘CACAGTGGGCCTGGGACTCAGGAGACTGTCCAGTGATCAAAGGCTTT
GG AGAATGCTGGG'I‘GGTCACAG GGGCCTGGGACTCAGGAGACTGTCCAGTGA'I‘CAAAGGCTTT
Key: exon : italics
primers : bold differences between RHD and RHCE in exon : underlined
SNPs for variant RHD alleles : double und rlined ?rst and last nucleotide of exon are
Consensus sequence is shown underneath each set of RHCE/RHD sequence.
Patent Application Publication
Jul. 23, 2009 Sheet 12 0f 26
US 2009/0186340 A1
Fig 21 Exon 8
RHCE : CAGGAGAGAGAGAGAAAACTGGAGGCTCTGAGAGGTTAAAGGACTTG RHD : CAG—GAGAGAGAGAGAAAACTGGAGGCTCTGAGAGGTTGAGGGACTTG
CAG
GAGAGAGAGAGAAAACTGGAGGCTCTGAGAGGTT A GGAC'I'TG
RHCE : CCCAGGGTCTTGCAGCTAGTAAGTGACAGAGCTGGGACTTGAGCTTGGGTTTTCTGACTCCTGGT RHD : CCCAGGGTCTTGCAGCTAGTAAGTGACAGAGCTGGGACTTGAGCTTGGGTTTTCTGAC'I‘CCTGGT
CCCAGGGTCT'I‘GCAGCTAGTAAGTGACAGAGCTGGGACTTGAGCTTGGGTTT'I‘CTGACTCCTGGT RJ-ICE' : CTGGTTCATTATCCATGAGGTGCTGGGAACTAAAATAAGCCACAATCTTGGAATCTCCGTCGCCT RHD
: CTGGTTCATTATCCATGAGGTGCTGGGAACTAAAATAAGCCACAATCTTGGAATCTCCGTCGCCT
CTGGTTCATTATCCATGAGGTGCTGGGAACTAAAATAAGCCACAATCTTGGAATCTCCGTC'GCCT RHCE : CCCTCCCTCCCACATGTCTGCG‘I'GGCTTTTTGGGAAAATGCCAGGGGAATGTACCAGCCAGGGAG RHD : CCCTCCCTCCCACATGTCTGCGTGGCTTTTTGGGAAAATGCCAGGGGAATGTACCAGCCAGGGAG
CCCTCCCTCCCACATGTCTGCGTGGCTTTT‘I‘GGGAAAATGCCAGGGGAATGTACCAGCCAGGGAG RHCE : AGGACCCTTGTTTTCCTCATGGCCCTTCCTGGCAATGGCACTACTGACACCGACAGTCCTTTTTG RHD
: AGGACCCTTGTTTTCCTCATGGCCCTTCCTGGCAATGGCACTACTGACACCGACAGTCCTTTTTG
AGGACCCTTGTTTTCCTCATGGCCCTTCCTGGCAATGGCACTAC'TGACACCGACAGTCCTTTTTG RHCE : TCCC'I‘GATGACCTC'I‘GCTGCCTGATGCCCAAGTGACCACCTCTGCTTTGTCATT’I‘CTAGGATTGG
RHD
: 'I‘CCCTGATGACCTCTGCTGCCTGATGCCCAAGTGACCACCTCTGCTT'I‘GTCATTTCTAGQLATTGG
TCCCTGATGACCTCTGCTGCCTGATGCCCAAGTGACCACCTCTGC'I'TTGTCATTTCTAGGATTGG RHC'E : CTTCCAGGTCCTCCTCAGCATTGGG'GAACTCAGCTTGGCC'ATCGTGATAGCTCTCAIGTCTGGTC RHD : CTTCCAGGTCCTCCTCAGCATTGGGGAACTCAGCTTGGCCATC’GTG'ATAGC'TCTCAQGTCTGGTC
CTTCC'AGGTCCTCCTCAGCATTGGGGAACTCAGCTTGGCCATCGTGATAGCTCTCA GTCTGGTC RHCE :
RHD
TCCTGACAGGTCAGTGTGAGGCCACC‘I‘TTCTTCCACCATTGCCAGGACACAGCACCCACGTC'CAG
: TC‘CTGACKQGTCAGTGTGAGGCCACCTTTCTTCCACCATTGCCAGGACACAGCACCCACGTCCAG
TCCTGACAGGTCAGTGTGAGGCCACCTTTCTTCCACCATTGCCAGGACACAGCACCCACGTCCAG
Key: exon : italics
primers : bold differences between RHD and RHCE in exon : underlined SNPs for variant RHD alleles : double underlined ?rst and last nucleotide of exon are
"
Consensus sequence is shown underneath each set of RHCE/RHD sequence.
Forward primer has 2 RHD speci?c nucleotides including one at the 3’ end. Reverse primer has universal sequence.
Patent Application Publication
Jul. 23, 2009 Sheet 13 0f 26
US 2009/0186340 A1
Fig. 21 Exon 9
RHCE : GAAAAAGGATTTCTGTTGAGACACTGTCGTTT’I'GACACACACAATATTTTGATTAATCTTGAGAT RHD : GAAAAAGGATTTC'I‘GTTGAGATACTGTCGT'I'T‘I‘GACACACA- —ATATTTCGATTAATCTTGAGAT
GAAAAAGGATT'I'CTGTTGAGA ACTGTCGTTTTGACACACA
ATATTT GATTAATCT'I‘GAGAT
RHCE : TAAAAATCCTGTGCTCCAAATCTTTTAACATTAAATTATGCATTTAAACAGGTTTGCTCCTAAAT RHD : TAAAAATCCTGTGCTCCAAATCTTTTAACA'I‘TAAAT'I‘ATGCATTTAAACAGQTTTGCTCCTAAAT
TAAAAATCCTGTGCTCCAAATCTTTTAACA'I'TAAATTATGCATTTAAACAGGTTTGCTCC'I‘AAAT RHCE : CTQAAAATATGGAAAGC'ACC'TCATGIGGCTAAATATTTTGATGAC'C'AAGTTTTCTGGAAGGTAAG RHD : CTEAAAATATGGAAAGCACCTCATGéGGCTAAATATTTTGATGACCAAGTTTTC'TGGAAQGTAAG CT AAAATATGGAAAGCACCTCATG GGCTAAATATTTTGATGACCAAGTTTTC'I‘GGAAGGTAAG RHCE : ATTTTTCACCTA'I‘TAACGTGATAGATT'I'TGAGTGCATGAACTTAAAAACATACCTGGGTATATAT RHD : ATT'I‘TTCACCTATTAACGTGATAGATTTTGAGTGCATGAACTTAAAAACATACCTGAGTATATAT
ATTTTTCACCTATTAACGTGATAGATT'I'TGAGTGCATGAACTTAAAAACATACCTG GTATATAT RHCE : GTTGACTTGCTGTTTATGAGTAAAACAAAAACAAAAATGGAGTAAGGAGCATTGCAGGAGGAACT RHD : GTTGACTTGCTGTTTATGAGTAAAACAAAAACAAAAATGGAGTAAGGAGCATTGCAGGAGGAACT
GT'I‘GACTTGCTGTTTATGAGTAAAACAAAAACAAAAATGGAGTAAGGAGCATTGCAGGAGGAACT RHCE : AGAGGAGAAACAAATCCATGATATGCATGTGTGTGGGGGAGGGTGGCGGGGAGGTGGTAAAGGTC RHD : AGAGGAGAAACAAATCCATGATATGCATGTGTGTGGGGGAGGGTGGCGGGGAGGTGGTAAAGGTC
AGAGGAGAAACAAATCCATGATATGCATGTGTGTGGGGGAGG‘GTGGCGGGGAGGTGGTAAAGGTC RHCE : ACCATTTCCCTGATACCTCAAATTCATTCAGAGTCAGGGATGAGACAGCTTTCACTGGCCACACT RHD : ACCATTTCCCTGATACCTCAAATTCATTCAGAGTCAGGGATGAGACAGCTTTCACTGGCCACACT
ACCATTTCCCTGATACCTCAAATTCATTCAGAGTCAGGGATGAGACAGCTTTCACTGGCCACACT RHCE : TCCCCTCCCGCTATCTGCAGTCCTCAGCGTAGCCAAATAGTTTGACATGCGGGTGACAGAACCCC RHD : 'I‘CCCCTCCCCCTATCTGCAGTCCTCAGCGTAGCCAAATAGTCTGACATGCGGGTGACAGAACCCC
TCCCCTCCC CTA'I'CTGCAGTCCTCAGCGTAGCCAAATAGT TGACATGCGGGTGACAGAACCCC
Key: exon : italics
primers : bold differences between RHD and RHCE in exon : underlined SNPs for variant RHD alleles : double underlined
Consensus sequence is shown underneath each set of RHCE/RHD sequence.
Patent Application Publication
Jul. 23, 2009 Sheet 14 0f 26
US 2009/0186340 A1
Fig 2K Exon 10
RHCE RHD
= CTGTT'I'CAAGAG'ATCAAGCCAAAATCAGTATGTGGGTTCATCTGCAATAAAAATGTTTGTTTTG : CTGTTTCAAGAGATCAAGCCAAAATCAGTATGTGGGTTCATCTGCAATAAAAATGTTTGTTTTG
CTGTTTCAAGAGATCAAGCCAAAATCAGTATGTGGGTTCATCTGCAATAAAAATGTTTGTTTTG RHCE RHD
: CTTTTACAGTTTCCTCATTTGGCTGTTGGATTTTAAGCAAAAGCATCCAAGAAAAACAAGGCCT : CTTTTACAGi-ZQZTTCCTCATTTGGCTGTTGGATTEQI‘AAGCAAAAGCATCCAAGAAAAACAAGGCCT
RHCE RHD
: GTTCAAAAACAAGACAACTTCCTCTCACTGTTGCCTGCATTTGTACGTGAGAAACGCTCATGAC : GTTCAAAAACAAGACAACTTCCTCTCACTGTTGCCTGCATTTGTACGTGAGAAACGCTCATGAC
CTTTTACAGTTTCCTCATTTGGCTGTTGGATTTTAAGCAAAAGCATCCAAGAAAAACAAGGCCT
GTTCAAAAACAAGACAACTTCCTCTCACTGTTGCCTGCATTTGTACGTGAGAAACGCTCATGAC RHCE
: AGCAAAGTCTCCT - TATGTATAATGAAACAAG- GTCAGAGACAGATTTGATATTAAAAAATTAA
RHD
:
AGCAAAGTCTCCAATGTTCGCGCAGGCACTGGAGTCAGAGAAAA- — -TGGAGTTGAATCCTTTC
AGCAAAGTCTCC
RHCE RHD
G
AC
G GTCAGAGA A
TG
TT AA
TT
: AGACTAAAAACTTAGTTTAAGAGTCA-—ATTTAATAAG-—-TTTAAAATA-AATGTTTAGTTTC : TCTGCCACTCTTTGAGGAGAATCTCACCATTTATTATGCACTGTAGAATACAACAATAAAATAC A
RHCE RHD
T T
TT
A
TCA
ATTTA TA G
T TA AATA AA
T A
T C
: ATTAGGATGATGC-TATCAATATTTTCTTGGTTA-CAGAC-ACATTATTAAAGTTTTGGGTTAA : AGCCA‘I'GTACCACATAACAACATCTTGGTAAACAACAGACTGCATATATGATGGTGGTCATCCA A T CTACAAATTT T ACAGAC CAT TAGT TA
Key: exon : italics
primers : bold ?rst and last nucleotide of exon are ‘sis
m u‘
Consensus sequence is shown underneath each set of RHCE/RHD sequence.
Patent Application Publication
Jul. 23, 2009 Sheet 16 0f 26
US 2009/0186340 A1
Figure 4
RHD Primer
Volume
ZOpM stock ([11) lOlF
1 .25
198R
1 .25
intlF
3 .5
297R.
3 .5
303B‘
1 .25
397R
1 .25
403E
l .25
499R
1.25
502B‘
1 .5
5Aluint4F
1 .5
599R
1 .5
6011?
1 .25
697R
1 .25
702]?
l .25
799R
1 .25
801E‘
l .5
899R
1 .5
901F
1 .1
998R
1 .1
IOmM Tris pI-IB
20.8
Total
50
Patent Application Publication
Jul. 23, 2009 Sheet 19 0f 26
US 2009/0186340 A1
Figure 6 ‘i:
‘I:
1% 2 % mmz a. a; 05 (BI
H cu g o
bp
1302
exon
FIN L 0
a a: g. a:
2 exon 5 RoHar- : 838bp
578 525
7 "—_-~—---
49s 470
8 9 1 5
415
6 -—---"""
407 304 256
3 4
RposHi'DrveRpoHsiDT veRngHa’DrieRvarH'inD’r