1. 1
Detection of Alu insertion at the PV92 locus of human chromosome16 using
PCR and gel electrophoresis
Nathan Cash, University of the Sunshine Coast
Word count [1545]
Abstract
Aluelementsare shortinterspersednuclearelementswithinmammaliangenomesthathave the
abilitytoretro-transpose.These retrotransposonsmake upasubstantial amountof the human
genome todayandhave beenimplicatedindifferentdiseasesandgeneticdisorders.Thisexperiment
usesPCRamplificationand agarose gel electrophoresisforthe detectionof anAluinsertionatthe
PV92 locusof humanchromosome 16. Conductedatthe Universityof the Sunshine Coastwitha
sample size n=10, resultsshowedthat8subjectstestedwere homozygousnegative and 1subject
was homozygouspositiveforanAluinsertionatthe PV92 locus.There wasinsufficientdatato
successfullyandaccuratelydetermine allelicfrequencyof the PV92Aluinsertion.
Introduction
Non-codingregionsmake upasubstantial amountof the humangenome.Althoughmuchof these
non-codingregionsare not transcribed,theyhave beenshowntohave some role inthe evolutionof
the humangenome and are the target forinsertionby mobile geneticelementsor‘transposable
elements’,whichincludethatof Aluelements. Aluelementsare transposable elementscommonin
primate andhuman genomes (1).These transposableelements are classifiedasshortinterspersed
nuclearelements(SINEs).Aluelements are furtherdefinedas retrotransposons,becauseof the RNA
intermediate thatisformedduringtranspositionwithinagenome. Alusequences are relativelysmall
usuallybeing300bpand make up 10.6% of the human genome withapproximately1millioncopies
throughout(diploid) (2-4).
Aluelements are thoughttobe derivedfrom7SLRNA,although Aluelementsdonotactively
participate inthe formationof signal recognitionparticlestowhich7SL RNA does (1).Aluinsertions
inparticularareas of the humangenome have beenlinkedtomany diseases,suchas manydifferent
formsof cancersand geneticdiseases (5).Aluinsertionsatthe PV92locusof humanchromosome 16
have beenusedasa markerinpopulationgeneticstoshow certainmigrationevents, andAlu
insertionselsewhere inthe genome have beenusedtoshow speciationandevolutionaryevents
such as that of the divergence of humanandprimate lineages(6,7).In humanchromosome 16, the

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Aluinsertionatthe PV92 locusisdimorphic,meaningthere canbe twoallelicvariations,either
homozygous(+/+),orheterozygous (+/-).Since thisparticularAluinsertionisnotfoundinall people
(homozygous-/-)itisthoughttoof occurredrecently ∽1-3 millionyearsago(mya) (8).Inthis
experimentthe presence of Aluatthe PV92 locuswas testedamonga small sub-populationof
Universitystudentstoanalyse the allelicvariancesandfrequencywithinthe classviaPCR
amplificationmethods.
Materialsand Methods
DNA samplepreparation and PCR
The procedure of DNA extractionwasperformedonstudentsatthe Universityof the Sunshine
Coast,and includedsamplesfrombothmalesandfemalesof variousage groups (n=30).DNA
samplescollectedforAlu insertiondetectionwere collectedfromcheek cells. Subjectsrinsedtheir
mouthswitha 0.9% saline solutionfor30secondswhilechewingthe sidesof theircheeks toloosen
cellsintothe solution.ThissolutionwascollectedandpreparedforPCR.Approximately20µl of
pelletfromeachsample wastransferredintotubes containingachelating(Instagene Chelex ion
exchange resin) age inhibitingthe activityof DNaseswhichmaybreakdownthe sample DNA.The
sampleswere incubatedat56˚C for 10mins to loosenconnective tissue,andthenat95˚C for5mins
to rupture cell membranesanddenature proteins.Aftercentrifugation 20µl of each DNA sample
were transferredintoPCRtubesanda PCR ‘mastermix’,aswell asforwardandreverse primersfor
the Alu allele,were added.Sampleswere placedinathermal cyclerforPCR(40 cycles).
AgarosegelElectrophoresis
Once PCR cyclingwascompleted,PCRgenomicDNA sampleswere storedfor2weeksbefore
electrophoresisandanalysis.A 1%agarose gel submergedinaTAE bufferwasusedtorun the gDNA
samples.Loadingbuffer(10µl) wasaddedto eachPCR sample andthe sampleswere loaded ontothe
agarose gel, whichwas run at 80V for30mins. Once complete,gelswere visualisedusinganUV
Transluminator(figure1).
Results
Little tono DNA was seeninthe agarose gelsafterPCR amplificationandelectrophoresisof the PV92
locus.The absence of DNA after electrophoresismeant analysiscouldnotoccur.For the purpose of
thispaperthe data from the MBT352 class of 2015 were used.Seenon(figure 1) a 13 lane agarose
gel wasrun, withlanes1, 9 and 13 containingDNA laddersize markers.Lanes2-8and 10-12
containedPCRproductsamples.The redcolourseenon (figure 1) showsthe amountof DNA

3. 3
present;thisindicatorisirrelevanttothisparticular analysis.The DNA ladderinlane 13is labelledat
size bands900bp and 600bp.
Figure 1. Amplified PCR products for the PV92 locus of human chromosome 16. DNA PCR products were run
using1% agarosegel and electrophoresis.Samples were run at80V for 30mins usinga horizontal gel
electrophoresis chamber containingTAE buffer solution.
Discussion
Background
Aluelementsare namedassuchfromthe activityof the restrictionendonuclease isolatedfrom
Arthrobacterluteus (Alu) thatrecognisesAlu repeatsequences(8).The insertionatthe PV92 locusof
humanchromosome 16 is classified underthe AluYsubfamily,the youngestsubfamilyof Alu
elements,presumablybeinginsertedintothe humangenome ∽1mya(9).Itis hypothesisedthatthe
mastergene forAlu elements comesfromthatof the 7SL RNA gene,whichencodesthe RNA portion
of the signal recognitionparticle involvedin translation-translocation(10,11). Overtime,mutations
inthismaster gene causedthe 7SL RNA gene to become atransposable elementwiththe Alu
elementloosingfunctionalitywithinthe genome(7).Aluelementsare consideredas‘parasitic
transposons’astheydo not encode theirownreverse transcriptase, integrase orendonuclease
requiredforcomplete transposition(8),insteadthey‘hijack’ the requiredenzymesthatare
producedfromothertransposons.Inthe the case of Aluretrotransposons,theyrelyonthe enzymes
producedbyLong interspersednuclearelements(LINEs),whichare anotherabundantclassof
retrotransposonswithin mammaliangenomes(12).The structure of Aluelementsconsists of two

4. 4
similarpartA and part B monomerswithanA5TACA6 linkerregionandapoly-A tail proceedingpart
B. The 5’ part A monomercontainspromotersequencesforRNA polymeraseIII (4).The variances in
the two monomersgive rise tothe differentsubfamiliesof Alu elements,all of whichare ∽300bp in
length(9).
PV92 locus
PV92 Aluinsertionhas noevidence supportinganycorrelationwithhumandisease.PV92Aluis in
the youngestclassof Aluelements(AluY) andhasshown tobe humanspecific(5).While this
experimentdidnotobtainsufficientdatatomake any conclusionsasto allelicfrequenciesor
variancesinthe populationtested,there isadequateliterature showingthe allelicfrequencyof the
PV92 Aluinsertionamongmanydifferentpopulations. A reasonastowhy the original resultswere
inconclusivecouldbe because of possible chelatingagentbeing transferred withthe productstobe
amplifiedviaPCR,ultimatelypreventinganyPCRamplificationtooccur.The allelicfrequency from
one studyof 715 subjectsacross31 worldpopulationsshowedthe AfricanPV92allelicfrequencyto
be 0.31 andthe EuropeanallelicfrequencyatPV92 to be 0.23 (13). Thisdata consistedof onlythe
presence orabsence of the Aluinsertat thislocus,anddidnot show differencesinzygosity (13).In
the experimentconductedaPV92locuscontaininganAluinsertwasexpectedtobe 941bp inlength
and a PV92 locusabsentof an Aluinserttobe 641bp in length,meaning heterozygous samples
wouldcontain twodistinctbands.Fromfigure 1 of the resultsitcan be seenthatsamplesinlanes2-
8, and 10 are homozygousnegativeforthe Aluinsertionatthe PV92 locus,while the DNA sample in
lane 12 isseento be homozygouspositive.The sampleruninlane 11 seemstohave possibly lacked
an adequate amountof DNA to accuratelyinterpretthe result.
Population geneticsand migration
WhencomparingAluinsertionsatspecificloci acrossa vast range of ethnicitiesandpopulations,
significantdifferencescanbe seen.There seemstohave beenloss’sincertainAluinsertionsinthe
humangenome vianatural selectionwhencomparingpopulationson eitherside of aprevious
populationbottle neck,suchthatoccurredwhenhumansmigratedoutof Africainto Europe (6,14).
Althoughthere are noknowngeneticeffectsof manyAluinsertions,inferencescanbe made as to
the originof a populationdue to the presence orabsence of Aluinsertions atparticularloci (14).
Alu elementsand implicationswith human disease
Althoughinsertionsof Aluelementsatthe PV92 locushave notbeenshowntobe implicatedwith
any diseasesordisorders,manyAlu insertionsinotherlocationsof the humangenome have been
shownto directlycause diseaseorhave a strongcorrelationwithcertaindiseases. Aluelements

5. 5
withinthe humangenome are passedontoprogenyina Mendelian fashion.Mostallelesare
inactive withinthe humangenomebutsome Alu elementsare still able totranspose,with
movementsof Aluelementsseenin 1outof every200 births(8, 3).The literature showsthat Alu
insertion (AluYa5) atcertainintronsof the NF-1 gene withinhumanchromosome17 can cause a
downstreamexondeletionduringintronsplicingcausingthe mutationresponsible for
neurofibromatosis type-1(15,2). Ina similarfashion,whenanAluinsertiontakesplace neara
certaingene or evenwithinthe coding regionof agene, itcan cause frame-shiftmutations. AnAlu
insertionatthe BRCA-2gene has beenshowntoincrease tumourformationandsubsequentlybreast
cancer (16, 17, 2). The BRCA genesare tumoursupressinggenes,sowhenmutationsoccur,their
productsbecome dysfunctional.
In conclusionourresultsdemonstratedthatAlupresence couldbe seeninthe populationtested,
althoughthere wasinsufficientdatatocalculate allelicfrequencies.Amongthe populationtested
mostsubjectswere showntobe homozygousnegativeforthe PV92Aluinsertion.Aluinsertionsare
abundantinthe human genome andcan be implicatedingeneticdiseasesaswell asformsof
cancers.Through populationgeneticsAluinsertiondifferencescanbe seenamongdifferent
populationsandlossesof certaininsertionshave occurredduringcertainmigrationevents
throughouttime.
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