The document discusses amplification refractory mutation system polymerase chain reaction (ARMS-PCR), a technique for detecting single-nucleotide polymorphisms. It involves designing allele-specific primers that differ by a single base at the 3' end to selectively amplify either the normal or mutant allele. A key principle is introducing a mismatch between the primer and template DNA to alter annealing temperatures and prevent amplification of the non-complementary allele. ARMS-PCR allows accurate and rapid diagnosis of genetic diseases and is widely used for conditions like sickle cell anemia and thalassemia. However, it is limited to detecting known SNPs and requires an internal control to avoid false results.

1. ARMS PCR

2. CONTENTS
Introduction
Principle
ARMS Primer designer
Procedure
Importance of PCR in the Diagnosis of Genetic
Disease
Advantages of ARMS PCR
Limitations of ARMS PCR

3. INTRODUCTION
The allele-specificPCRalsocalledasan ARMS- PCR (amplification refractory mutation system) orPASA
(PCRamplification of specificalleles) orAS-PCRusedtodetectthe SNPs. More specifically, itis
adopted todetectthe known SNPs(single nucleotide polymorphism).
The term suggeststhatthe technique usedinthistype of PCRisspecifictothe particulartype of allele.
An allele isthe alternativeformof a gene. If one allele hasanSNPandthe otheralternative formis
normal,we can analyse boththe allelesbydesigningspecificprimersforeachallele.Forthat,we have
to modifythe single base atthe 3’ endof the primer(one primermatchesthe normal allele andone
primermatchesthe mutantallele).
The PCR is performedsimultaneouslyinasingle reaction.If amutantallele ispresent,thenthe PCR
amplifiesthe mutantallele orif the normal allele ispresent,the normal allelewill amplify.
AmplificationRefractoryMutationSystem(ARMS) isanapplicationof PCRinwhichDNA isamplifiedby
allele specificprimers.InPCRmismatchatthe 3’ endof the primercan dramaticallyreduce the
annealingandhence the amplification.Thisisdue tothe absence of 3’ to 5’ exonuclease proofreading
activityof Taq polymerase.HighfidelityDNA polymerases,thathave thisactivity,cannotbe usedin
ARMS.
It isan extremelyusefulmethodforidentificationof pointmutationsorpolymorphisms.Since the
ARMS PCR ismostlydone toidentifyamutationora polymorphismitisalsoimportantthatitshould
be able to identifywhetherthe change inDNA isheterozygousorhomozygous.A heterozygoteor
homozygote isdifferentiatedbyusingARMSprimersforthe mutant/polymorphicandthe normal (wild
type) alleles.The reactionsforthe mutantandthe normal allelesare usuallycarriedoutinseparate
tubes.Butthese maybe done inthe same tube after labellingthe twoprimerswithdifferent
fluorescentdyes.
Kary Mullis described the technique of in vitro amplification inthe year1983. Aftera few yearsof the
discovery of the actual PCR technique, C. R. Newton and co-workers discovered the ARMS-PCRor
allele-specificPCRtechnique.
The technique ismajorlyused forthe genotyping of the single nucleotide polymorphismwiththe help
of the refractoryprimers.
ARMS-PCR,amplificationrefractorymutationsystempolymerase chainreaction

4. PRINCIPLE
The mechanismof the ARMS PCR isbasedon the modification of the primersfordifferentalleles.
Here, the 3’ end of the primers ismodified insuchaway that one setof the primercan amplify the
normal allele andothers canamplify the mutantallele. The mismatchsingle base isintroduced atthe
3’ endof the primer. Thismismatchallowsthe primertoamplify one single allele.
The concept of mismatch:
Here the mismatchbetween the primerandthe template DNA playsacrucial role inachievingthe
amplification.
Introduction of a mismatchat the 3’ endof the primeraltersthe annealingtemperature forthat
particularallele.
Strong mismatch: G/A, C/T, T/T
Mediummismatch: A/A, G/G,C/C,
Weakmismatch: C/A, G/T
Due to the absence of the exonuclease activityof TaqDNA polymerase,the mismatchcannotbe
repaired.

5. ARMS PRIMER DESIGN
1. The ARMS PCRrequiresapair of primersincludingacommonandan ARMS primer.The
commonprimerislike anyotherPCR primer.Butthe ARMS primerhas the followingspecial
features:
2. The primeris usually30 basesin length.
3. The nucleotide atthe 3’ endof the primershouldbe complementarytothe targetnucleotide
i.e., G forC or C forG and T for A or A forT. Mismatch at thispositioncandramaticallyreduce
the amplification. A:G,G: A,and C:C mismatcheshave the worsteffectwhereasthe other
mismatcheshave varyingdegreesof effect.For example, inamutationwithA-Tsubstitution
the ARMS primerfor the mutantallele shouldhave the lastnucleotide complementarytothe
nucleotide Ti.e., itshouldhave A.The primerforthe normal allele atthe same positionshould
be complementarytothe nucleotide A i.e., itshouldhave Tasshowninthe figure below.
4. An additional mismatchatone of the lastfive nucleotidesof the ARMSprimerfurther
increasesits specificity.
5. It iscustomaryto include aninternal PCRcontrol inARMS reactions.A pair of primersis
designed toamplifyaregionof the gene of interestthatusuallyisfree of mutations.An
amplificationof the internal control regionandnoamplificationbythe ARMSprimerindicate a
true negative.Ina false negativeresultneitherthe ARMSprimernorthe internal control
showsanyamplification.There couldbe several reasons for the false negative result e.g., too
little or too much DNA, poorqualityof DNA template,failure toaddprimer,Taq,or other
reagentsandpresence of PCRinhibitors.
6. The sensitivityandspecificityof anARMS reactioncanbe controlledbystringentreaction
conditions.Goodprimerdesign,higherannealingtemperature andlimitednumberof cycles
are importantinavoidingfalse results.The numberof cyclesshouldbe justenoughtogive a
clearpositive result.Increasingthe numberof cyclesun-necessarilycancause false positives.
The usual lengthof ARMS primeris30 bases.Primersof thislengthhave ahighTm and
annealingtemperature andare therefore more specific.

6. ARMS primersforthe normal and the mutant allele of apointmutation,IVSI-5(G-C),inthe β-globin
gene.SegmentA showsthe matchedARMSprimerforthe mutation(C),segmentBshowsthe
mismatchbetweenthe mutantARMSprimerandthe normal sequence (G), segmentCshowsthe
matchedARMS primerforthe normal sequence (G),segmentDshowsthe mismatchbetweenthe
normal ARMS primerandthe mutation(C).A deliberate mismatch(T:T) isalsoaddedat position
enclosedbyblue box.
PCR conditions for ARMS
Locus: β-globingene
GenBank accession NG_000007.3
Allele: β-thalassemiamutationIVSI-5(G-C)
o Forward primer: 5’-ACCTCACCCTGTGGAGCCAC
o Reverse primer(ARMS)
5’-CTCCTTAAACCTGTCTTGTAACCTTGTTAG
o Amplified product 285 bp
o Control primer(forward):
5'-CAATGTATCATGCCTCTTTGCACC
o Control primer(reverse):
5'-GAGTCAAGGCTGAGAGATGCAGGA
o Amplified product: 861 bp
 Reaction volume: 25μl
 PCR mix: 22μl
 Primerconcentration: 1μl (5 p mol each/ μl) (Chapter3)
 Taq polymerase: 0.5 units(0.1μl)
 Template DNA: 2μl (~300ng)
Thermal cycling:
o Initial denaturation: 1 minute at94o
C
o No. of cycles: 25
o Denaturation: 1 minute at94o
C
o Annealing: 1 minute at65o
C
o Extension: 1 minute 30 secondsat 72o
C
o Final extension: 3 minutesat72o
C
Electrophoresis: 10 X 10 cm 6% polyacrylamide,40minutesat150 volts.
Staining: 0.1% silvernitrate
Result: below

7. Silver-stainedpolyacrylamidegel electrophoresisafterARMSPCR.All lanesshow 861bp internal
control fragment.Lanes1-3 shows285bp fragmentof IVS1-5 mutation.Lanes4-5 are negative forthe
same mutations.Lane 7 showsallelicladderforvariousthalassaemiamutations.
PROCEDURE
The process of the ARMS PCR issimple andvery effective. Noharmful radiolabellingisinvolved inthe
ARMS allele-specificPCR.
The procedure of ARMS PCR isdivided into4steps:
1. Primerdesigning
2. Amplification
3. Agarose gel electrophoresis
4. Resultsinterpretations
PRIMER DESIGNING FOR ARMS PCR:
The primermust be allele-specific.
Suppose ourDNA sequence hasG-A pointmutation viz, Gin normal allele andA inplace of G inthe
mutantallele.
We have to designaforwardprimerinsuch a mannerthat for normal allele the primercontains C
(complementary toG) at 3’ endand the mutantprimercontains T in place of C.
The magic of the presenttechniquehashappened when we addamismatchbase nearto our SNP at
the 3’ end.
The mismatchis the keyfactor inachievingthe amplification. If the mismatchisweakthe chance of
amplification ishigher. Addstrongmismatchnearthe 3’ endof the primer(at -2 position ideally)
hence, inthe non-complementary alleleitcannotbindtothe DNA sequences andtherefore terminates
amplification.
C: T, G: A andA: G are the strongmismatchbase pairswhichreduce the amplification process upto
100-fold.The reverse primeroranotherprimer(which isnotmodified) generally remains the same.
The primerlengthof the allele-specificprimermustbe between 20 to 30 nucleotides.

8. Once our primerisready, we have to modify the amplification conditions forachievinghigher
amplification.
AMPLIFICATION CONDITIONS:
The annealingtemperatureshould higher(donotcompromise the annealingtemperature).
The concentration of the PCR components isgivenintothe table below,
The PCR reaction preparation recipe.
Importantly,the PCRcyclesforARMS-PCRare lowerthanthe normal PCR reaction.Seta PCRcycle
between22to 25 but not 35. Asthe PCR cyclesincrease the chance of false-positive resultsincreases.
“An additional mismatchtothe 3’ endafterthe firstmismatchincreasesthe specificityof the
ARMS-PCRreaction.”
In the ARMS PCR,the internal control playsa crucial role,itfacilitatesadditionalaccuracyinthe
allele-specificPCRbyreducingthe chance of the false-positive results.
AGAROSE GEL ELECTROPHORESIS:
The beauty of the ARMS PCR isthat it does notrequire anyhybridization steps. Whichwill make this
technique more suitable andmore reliable forthe diagnosis.
The amplified fragmentsare directly loadedonthe 2% agarose gel for gettingthe results. The samples
are loaded sequentially onthe agarose gel andrun for 45 minutes.

9. RESULTS AND INTERPRETATION:
First, observe the internal control. If the internal controls bandsare presentinall the wellsindicates
that our reactioniscompletelyfine, nofalse-positive resultsare present. Now analyseeachbandin
normal as well asinmutant allele. The graphical representation of the agarose gel electrophoresis
resultsisshown inthe figure below.Suppose we have three samplesone normal, one heterozygous
carrier andone homozygous dominant. We have prepared 2tubes foreach sample, atotal of 6 tubes
withone positive control andtwonegative control.

10. As shownintothe figure above,anonlysingle bandisobservedinthe homozygousnormal sample,
twobands (one fornormal and one formutant) are observedinthe heterozygouscarriersample anda
single bandof a mutantallele isobservedinthe homozygousdiseasecondition.The wells7,8 and 9
are the controls.
IMPORTANCE OF ARMS-PCR IN THE DIAGNOSIS OF GENETIC
DISEASE:
The ARMS PCRis one of the importanttoolsinthe geneticdisease diagnosis inrecentdays. The
restriction digestion methodisnot100% accurate and also, not all the sequences have the restriction
site.
Therefore,restrictiondigestionisnotapplicableinall typesof mutationorpolymorphism.
The allele-specificPCRisan accurate methodforsingle-gene disordershavingthe SNPs.Itisalsoa gold
standardmethodforSickle cell anaemiaandthalassemialikeinherited disorder.
Also,itisapplicable inmutationdetectionof JAK2and HIV.Commercial kitsare available forthe
differentrange of disorderssuchasHbS and JAK2gene SNPsdetectionkits.
ADVANTAGE OF ARMS PCR:
 The technique isexclusively forthe SNP genotyping.
 Further, homozygous andheterozygous canbe detected bythistechnique
 It iswidely applicable inthe single gene pointmutationdetection suchassickle cell anaemia
and thalassemia.
 It isfast, reliable accurate andrapid.
LIMITATION OF ARMS PCR:
 Deletion/othermajorduplication andchromosomal abnormalitiescannotbe detected.
 Only knownSNPsare detected byARMS-PCR.
 Internal control isrequired because of the chance of the false-negative results.
 It istemperature-sensitive. A minute fluctuationinthe temperatureleadstofalse-positive
results.
 Thousandsof SNPscannot be detectedinasingle assay.

11. CONCLUSION
The amplification-refractorymutationsystem(ARMS) isasimple methodfordetectinganymutation
involvingsinglebase changesorsmall deletions.ARMSisbasedonthe use of sequence-specificPCR
primersthatallowamplificationof testDNA onlywhenthe targetallele iscontainedwithinthe
sample.Followingan ARMS reaction, the presence orabsence of aPCR product isdiagnosticforthe
presence orabsence of the targetallele.
The amplification-refractorymutationsystem(ARMS) isasimple methodfordetectinganymutation
involvingsinglebase change.
The ARMS-PCRmethodispopularfor the detection of known SNPs;however, itisa very tedious and
time-consumingprocess toencountermore SNPsatonce. Because itis allele-specific, the accuracy
of ARMS-PCRishigher.
ARMS-PCR can be utilizedasacost-effective,rapidandreproduciblemethodforSNPgenotyping
especiallywhile performinglarge-scale epidemiological/associationatstudies.
Some of the deletions canalsobe screened usingARMS-PCRmethod.
REFERENCES
 Dr. Tushar Chauhan. 27Feb.2019, GeneticEducation.co.in
 Tushar Chauhan, Tushar Kachhadiya.19Aug.2018, Genetic.co.in
 Dr. Satyendra Kumar.30Dec.2013,Technology Health & Medicine
 Baoyu Peng. July 2017, reseachgate.net