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Protocol:GapPCRIthapedia

Protocol:GapPCR
FromIthapedia

Contents
1Principle
2Method
3Materials
4MultiplexGapPCR
4.1MultiplexPCRprotocolforthediagnosisof3.7and4.2deletions
4.2MultiplexPCRprotocolforthediagnosisoftheMEDand()20.5deletions
4.3MultiplexPCRprotocolforthediagnosisoftheSEA/FIL/THAIothalassaemiadeletions
4.4PCRprotocolforthediagnosisofthe(anti3.7)allele
4.4.1ReactionMix1
4.4.2ReactionMix2
4.4.3Interpretationoftheresultsofreactionmixes1&2forthediagnosisofthe(anti3.7)allele
5References

Principle
GenedeletionmutationsintheglobingeneclustermaybedetectedbyPCRusingtwoprimerscomplementarytothesenseandantisense
strandintheDNAregionsthatflankthedeletion.Forsmalldeletionsoflessthanapproximately1kb,theprimerpairwillgeneratetwo
products,thesmallerfragmentarisingfromthedeletionallele.Forlargedeletions,thedistancebetweenthetwoflankingprimersistoogreatto
amplifythenormalalleleandproductisonlyobtainedfromthedeletionallele.Inthesecasesthenormalalleleisdetectedbyamplifyingacross
oneofthebreakpoints,usingaprimercomplimentarytothedeletedsequenceandonecomplimentarytotheflankingDNA.
GapPCRisusedtodiagnosesomethlassaemiadeletions,HPFHdeletions,thalassaemiadeletions(Table1)andalsothetriplegene
locusgeneratedbythe3.7kbsinglegenedeletion[5].AtypicalgapPCRtestisillustratedinFigures1and2.Forthediagnosisof
thalassaemia,theprimerscannowbemultiplexed.The3.7kband4.2kb+ thalassaemiadeletionscanbedetectedinoneassay(Table2),the
MEDand()20.5othalassaemiadeletionsinoneassay(table3)andthe3SoutheastAsianothalassaemiadeletionsinoneassay(Table4).
TheprotocolsusedintheOxfordlaboratoryforthemultiplexingoftheseprimersaregiveninTables24,butitshouldbenotedthatthe
quantityofeachprimerpairrelativetotheothersmayneedadjustmenttogainoptimumamplificationofalltheproducts.PCRdiagnosisofthe
triplegene(anti3.7allele)requirestwoseparateassays(Tables5&6).Thepresenceofthealleleisdiagnosedfromacomparisonofthe
resultsofeachassayrunsidebysideandthegenotypeoftheDNAsamplecanbededuced(Table7)inmostinstances.
Table1ThalassaemiadeletionmutationswhichhavebeendiagnosedbygapPCR

othalassaemia

SEA
MED
()20.5
FIL
THAI

[6]
[6]
[6]
[8,9]
[8.9]

+ thalassaemia

3.7
4.2

[7]
[7]

othalassaemia

290bpdeletion
532bpdeletion
619bpdeletion
1393bpdeletion
1605bpdeletion
3.5kbdeletion
10.3kbdeletion
45kbdeletion

[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]

()othalassaemia

HbLepore
[20]
Spanish
[20]
Sicilian
[20]
Vietnamese
[20]
Macedonian/Turkish[20]

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(A)othalassaemia

Indian
Chinese

[20]
[20]

HPFH

HPFH1(African) [20]
HPFH2(Ghanaian) [20]
HPFH3(Indian)
[20]
HbKenya

Method
1.Setupthereactionmixturetoafinalvolumeof22lintoa0.5mltubewiththefollowingcomponentsasrequired:1lgenomicDNA
(100ng/l)(1lofforwardprimerflankingsequence(10pmol/l),1lreverseprimerflankingsequence(10pmol/l),1lofprimer
deletedsequence(10pmol/l),1lofprimerinvertedsequence(10pmol/l),2.5lof1.25mM(dNTPmixture),2.3lof10xGap
PCRbufferasrecommendedfortheprimersintheoriginalreference(seeTable5.2)andbelow.
2.Thebufferrecommendedforthethalassaemiaprimersis750mMTrisHClpH8.8,200mM(NH4)2SO4,0.1%Tween20).Thebuffer
forthethalassaemiaprimersshouldalsocontains0.5Mbetaineand0.5%DMSO(thiscanbeachievedbyadding2.5lof5Mbetaine
and1.25l10%DMSO).
3.Makeupallreactionstoafinalvolumeof22lbyaddingsteriledH2O.
4.Overlaywith25lofmineraloil.
5.Prepareenzymemixture:0.2lreactionbuffer(10x),0.1lAmpliTaq(5U/l)(PEBiosystems)forthegeneprimers,0.1lPlatinum
Taq(5U/l)(Invitrogen)forthegeneprimers,and2.7lsteriledH2O,tomake3l.
6.Mixenzymemixtureandholdonice.
7.Placereactionmixturesinthermalcyclerandperformonecycleasfollows,adding3loftheenzymemixafter2minutesofthe94oC
denaturationstep:4minat94oC/1minat5565oC(asrecommended)/1.5minat72oC
8.Continuefor33cycleswiththefollowingstepspercycle:1minat94oC/1minat5565oC(asrecommendedinthepublishedreferences
orintables5.95.13)/1.5minat72oC
9.Finishwithonecycleasfollow:1minat94oC/1minat5565oC(asrecommended)/10minat72oC.
10.Holdat15oCuntilgelelectrophoresis.
11.Removetubesfromthermalcycler.Add5lofbluedye,mixandcentrifuge.
12.Dependingonexpectedproductsizes,loada20laliquotontoa13%agarosegelandrunat100Vfor45minto2hrsin1xTris
borateEDTAbuffer(TBE).
13.Staingelinethidiumbromidesolution(0.5g/ml)for1530minutes,visualisebandsonaUVlightbox(312nm)andphotographwith
anelectroniccamerasystemoraPolaroidCU5camerafittedwithanorangefilter(e.g.Wratten22A).Forguidancereinterpretationsee
notes6,7and8.

Materials
1.DNTPs:Addtogether50lofa100mMsolutionofeachdNTP(aspurchased)and3.8mlofdistilledwater.The1.25mMdNTPstock
solutionshouldbestoredinfrozenaliquots.
2.10xGapPCRreactionbuffer(compositionvariesaccordingtoprimersused)seemethodsandTable2
3.Betaine(SigmaAldrichChemicalCoLtd,England)
4.MineralOiltooverlayPCRreactions
5.PCRprimers:dilutealiquotsofprimerstocksolutionstomakeaworkingsolutionof1ODunit/mlandstorefrozen.
6.Ammoniumsulphatebuffer:75mMTrisHCl(pH9.0),20mM(NH4)2SO4,2.0mMMgCl2,0.01%Tween20,10%DMSO,10mM
mercaptoethanol(allfinalconcentrations).
7.Taqpolymerasesand10xTaqbuffers:inmylaboratoryareasfollows,AmpliTaqGold(PEBiosystems)worksbestforARMSPCR/RE
digestionassaysandPlatinumTaq(GibcoLifeTechnologies)forgapPCR.
8.TrisborateEDTA(TBE)buffer:89mMTrisborate,89mMboricacid,10mMEDTA,pH8.0.
9.Bluerunningdye(15%ficoll/0.05%bromophenolblue).
10.UVtransilluminatorandPolaroidcamera,orUVelectroniccamerasystem
11.0.5g/lEthidiumbromide

MultiplexGapPCR
Specificprimerdetailsetcarelistedbelowforthemultiplexdiagnosisofthecommonthalassaemiagenotypesandthetriplicatedglobin
allele.Figures5.10and5.11showexampleresultsallthecommonthalassaemiageneotypes.

MultiplexPCRprotocolforthediagnosisof3.7and4.2deletions
1.Primersequences[1]
AnnealingtempoC
2/3.7F
CCCCTCGCCAAGTCCACCC
64
3.7/20.5R AAAGCACTCTAGGGTCCAGCG 64

primer description sequence

1
2

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3
4
5

Protocol:GapPCRIthapedia

2R
4.2R
4.2F

AGACCAGGAAGGGCCGGTG
64
CCCGTTGGATCTTCTCATTTCCC 64
GGTTTACCCATGTGGTGCCTC 64

2.PCRreactionmix
component
l
2/3.7F(10M)
1.0
2R(10M)
0.25
2/20.5R(10M)
1.0
4.2F(10M)
1.0
4.2R(10M)
1.5
10xbuffer(750mMTrisHClpH8.8,
200mM(NH4)2SO4,0.1%Tween20) 2.5
25mMMgCl2
dNTPs(1mM)
Betaine(5M)
DMSO(10%)
PlatinumTaq(5units/ml)
DNAtemplate(100ng/ml)
Water

1.5
5.0
3.75
1.25
0.1
1.0
5.2

3.Gelelectrophoresisconditions
RunPCRproductsouton1.5%(1:1Nusieve:agarose)gelfor23hours
4.Interpretationofresults
PCRFragmentsize(bp) Genotype
Productofprimers
2020
+ thalassaemia:3.7 1+2
1800
Normal()
1+3
+
4.2
1628
thalassaemia: 4+5

MultiplexPCRprotocolforthediagnosisoftheMEDand()20.5deletions
1.Primersequences[2]
AnnealingtempoC
MED(F) CGATGAGAACATAGTGAGCAGAATTGCAGG 60
MED(R) ACGCCGACGTTGCTGCCCAGCTTCTTCCAC
60
SEA(F)
CTCTGTGTTCTCAGTATTGGAGGGAAGGAG
60
SEA(N) TGAAGAGCCTGCAGGACCAGGTCAGTGACCG 60
()20.5(F) GGGCAAGCTGGTGGTGTTACACAGCAACTC 60
60
()20.5(R) CCACGCCCATGCCTGGCACGTTTGCTGACG

primer name
1
2
3
4
5
6

sequence

2.PCRreactionmix
component
SEA(F)(10M)
SEA(N)(10M)
MED(F)(10M)
MED(R)(10M)
()20.5(F)(10M)

l
1.0
0.5
0.4
0.4
0.4

0.4
()20.5(R)(10M)
10xbuffer(750mMTrisHClpH8.8,
2.5
200mM(NH4)2SO4,0.1%Tween20)
25mMMgCl2
1.5
dNTPs(1mM)
Betaine(5M)
DMSO(10%)
PlatinumTaq(5units/ml)
DNAtemplate(100ng/ml)
Water

4.0
3.75
1.25
0.1
1.0
6.2

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3.Gelelectrophoresisconditions
RunPCRproductsouton2%(1:1Nusieve:agarose)gelfor11.5hours
4.Interpretationofresults
PCRFragmentsize(bp) Genotype
Productofprimers
o
20.5
1175
5+6
thalassaemia:()
1010
875

Normal()
+ thalassaemia:MED

3+4
1+2

MultiplexPCRprotocolforthediagnosisoftheSEA/FIL/THAIothalassaemiadeletions
1.Primersequences[3]
AnnealingtempoC
FIL(F) AAGAGAATAAACCACCCAATTTTTAAATGGGCA 60
FIL(R) GAGATAATAACCTTTATCTGCCACATGTAGCAA 60
SEA(F) CTCTGTGTTCTCAGTATTGGAGGGAAGGAG
60
SEA(N) TGAAGAGCCTGCAGGACCAGGTCAGTGACCG 60
SEA(R) ATATATGGGTCTGGAAGTGTATCCCTCCCA
60
THAI(F) CACGAGTAAAACATCAAGTACACTCCAGCC
60
THAI(R) TGGATCTGCACCTCTGGGTAGGTTCTCTACC
60

primer name
1
2
3
4
5
6
7

Sequence

2.PCRreactionmix
component
l
SEA(F)(10M)
2.0
SEA(N)(10M)
1.0
SEA(R)(10M)
1.0
FIL(F)(10M)
4.0
FIL(R)(10M)
4.0
THAI(F)(10M)
1.0
THAI(R)(10M)
1.0
10xbuffer(750mMTrisHClpH8.8,
200mM(NH4)2SO4,0.1%Tween20) 2.5
25mMMgCl2
dNTPs(1mM)
Betaine(5M)
DMSO(10%)
PlatinumTaq(5units/ml)
DNAtemplate(100ng/ml)
Water

1.5
4.0
3.75
1.25
0.1
1.0
0.65

3.Gelelectrophoresisconditions
RunPCRproductsouton1.5%(1:1Nusieve:agarose)gelfor2hours
4.Interpretationofresults
PCRFragmentsize(bp) Genotype
Productofprimers
1010
Normal()
3+4
660
+ thalassaemia:SEA 3+5
550

+ thalassaemia:FIL

495

+ thalassaemia:THAI 6+7

1+2

PCRprotocolforthediagnosisofthe(anti3.7)allele
ReactionMix1
1.Primersequences
AnnealingtempoC
GATGCACCCACTGGACTCCT 55

primer description sequence

1

C10

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C3

CCATTGTTGGCACATTCCGG 55

2.PCRreactionmix
component
l
C10(10M)
1.0
C3(10M)
1.0
10xbuffer(750mMTrisHClpH8.8,
200mM(NH4)2SO4,0.1%Tween20) 2.5
25mMMgCl2
dNTPs(1mM)
Betaine(5M)
DMSO(10%)
PlatinumTaq(5units/ml)
DNAtemplate(100ng/ml)
Water

1.5
5.0
3.75
1.25
0.1
1.0
12.9

3.Gelelectrophoresisconditions
RunPCRproductsofreactionmixture1outon2%(1:1Nusieve:agarose)gelfor2hours,inlanenexttothoseofreactionmixture2.
SeeTable5.14forinterpretationofresults.
4.Productsizes
PCRFragmentsize(bp) Genotype
Productofprimers
+
3.7
Noproduct
thalassaemia: 1+2
1900
1900

Normal()
:(anti3.7)

1+2
1+2

ReactionMix2
1.Primersequences
AnnealingtempoC
GATGCACCCACTGGACTCCT 50
CCATGCTGGCACGTTTCTGA 50

primer Description sequence

1
2

C10
C2

2.PCRreactionmix
Component
l
C10(10M)
1.0
C2(10M)
1.0
10xbuffer(750mMTrisHClpH8.8,
2.5
200mM(NH4)2SO4,0.1%Tween20)
25mMMgCl2
1.5
dNTPs(1mM)
Betaine(5M)
DMSO(10%)
PlatinumTaq(5units/ml)
DNAtemplate(100ng/ml)
Water

5.0
3.75
1.25
0.1
1.0
12.9

3.Gelelectrophoresisconditions
RunPCRproductsofreactionmixture2outon2%(1:1Nusieve:agarose)gelfor2hours,inlanenexttothoseofreactionmixture1.
SeeTable5.14forinterpretationofresults.
4.Productsizes
PCRFragmentsize(bp) Genotype
Productofprimers
2100
Normal()
1+2
2100
:(anti3.7)
1+2
1900
+ thalassaemia:3.7 1+2
Interpretationoftheresultsofreactionmixes1&2forthediagnosisofthe(anti3.7)allele
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1.Products(bp)
Genotypes
/
3.7/

Primers:C2+C10 Primers:C3+C10
2100
1900
2100+1900
1900

3.7/3.7

1900

/3.7

2100+1900

/or/ 2100

2100+1900
2100+1900

Notes:
1.allele:amplifieswithC3+C10(1.9kb)andC2+C10(2.1kb).
2.The3.7allele:amplifieswithonlyC2+C10.Givesashorterproduct(1.9kb)thannormalbecauseofthedeletedgene.
3.Theallele:amplifieswithC3+C10(1.9kb)andtwicewithC2+C10(2.1kb)becauseoftheextragene.
2.Possiblegelpatterns
genotypes
/
/or/
3.7/
3.7/3.7
/3.7
Primerpairs C2+C10 C3+C10 C2+C10 C3+C10 C2+C10 C3+C10 C2+C10 C3+C10 C2+C10 C3+C0
____
____
____
____
____
____ 2.1kb
Bandpatterns
____
____
____
____
____
____
____ 1.9kb
Figure1.Thediagnosisof+ thalassaemiadeletionmutationsbymultiplexGAPPCRusingtheprimersdescribedunderPrimersequences
[1]above.

Figure2ThediagnosisofothalassaemiadeletionmutationsbymultiplexGAPPCRusingtheprimersdescribedunderPrimersequences[2]
&[3]above.

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