1he 1echnlcal Þrocess

of ulrecL-Lo-Consumer
CeneLlc 1esLlng

!" $"%&'()*+,- .-/0'+1*+&" %&'
2*34-"*/ +" *5- 6+%- 20+-"0-/

Laura 8ruce LnCL 202C 03/27/2014

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1he purpose of Lhls documenL ls Lo provlde undergraduaLe sLudenLs ln Lhe llfe sclences wlLh a
conLemporary, lnformaLlve descrlpLlon of Lhe Lechnlcal process behlnd dlrecL-Lo-consumer (u1C) geneLlc
LesLlng. An emphasls ls placed on Lhe progresslon of Lhe laboraLory and analyLlcal sLeps. Slnce Lhls
documenL does noL lnclude exLenslve molecular-level descrlpLlons, baslc background knowledge of
blochemlcal concepLs and laboraLory Lechnlques ln blology wlll be beneflclal for full comprehenslon.
1hus, whlle Lhls documenL can be useful Lo anyone who has an lnLeresL ln genomlcs, lL wlll be especlally
sulLed Lo sLudenLs sLudylng Lhe llfe sclences, who llkely have relevanL prlor knowledge ln cell blology,
blochemlsLry, and geneLlcs. noLably, Lhe lnformaLlon ln Lhls documenL ls lncreaslngly valuable, as Lhe
growlng popularlLy of u1C geneLlc LesLlng ls pressurlng Lhe healLhcare sysLem Lo move Lowards a more
personallzed sysLem. lL ls essenLlal LhaL sLudenLs pursulng careers ln Lhe llfe sclences are aware of
developmenLs ln bloLechnology, lncludlng u1C geneLlc LesLlng.

As more lndlvlduals become lnLeresLed ln whaL Lhelr genome may uncover, genet|c test|ng, a medlcal
screenlng process LhaL ldenLlfles varlaLlons ln an lndlvldual's chromosomes, genes, or proLelns, has
become lncreaslngly prevalenL. 1o some exLenL, geneLlc LesLlng has been rouLlnely used ln forenslcs,
genealogy, feLal and newborn screenlng, prenaLal plannlng, and dlsease dlagnosls. Cnly recenLly,
however, has geneLlc LesLlng become readlly avallable for lndlvlduals.
ln 2003, Lhe Puman Cenome Þro[ecL was compleLed, mapplng
Lhe enLlre human genome and sheddlng llghL on how our genes
lnfluence our physlology and reveal our ancesLral rooLs. As a
resulL, sclenLlsLs are lncreaslngly capable of undersLandlng Lhe
relaLlonshlps beLween genotype, Lhe molecular codlng of genes
for a parLlcular LralL, and phenotype, Lhe physlcal manlfesLaLlon
of LhaL parLlcular LralL, whlch can also be lmpacLed by
envlronmenLal facLors.

Cne speclflc way of measurlng genoLype and geneLlc varlaLlon
beLween lndlvlduals ls by analyzlng Lhelr s|ng|e nuc|eot|de
po|ymorph|sms (SNÞs). SnÞs are naLurally occurrlng muLaLlons
(changes) ln Lhe four posslble nucleoLlde bulldlng blocks of unA (C: cyLoslne, C: guanlne, A: adenlne, 1:
Lhymlne). As shown ln 7+83'- 9, ln Lhe hlghllghLed reglon of unA, Lhree lndlvlduals have Lhree dlfferenL
nucleoLldes, whlch represenL SnÞs. 1yplcally, an lndlvldual's genome wlll conLaln abouL 10 mllllon SnÞs,
mosL of whlch have no effecL. SnÞs only affecL phenoLype lf Lhey occur wlLhln a gene or lLs regulaLory
reglon. ÞhenoLyplcally, Lhese effecLs mlghL be llnked Lo LralLs llke eye color, lnherlLed condlLlons, or
Lendencles ln drug meLabollsm. SnÞs can also be used Lo Lrack ancesLry.
(Source: 1he 8road lnsLlLuLe,
I|gure 1. SnÞs

D|rect-to-consumer (D1C) genet|c test|ng ls a Lype of healLh and ancesLry geneLlc LesLlng LhaL ls sold
dlrecLly Lo lndlvlduals, wlLhouL lnpuL from healLhcare provlders or lnsurance companles. 1hls servlce
relles on a Lechnlcal process LhaL accesses and analyzes all of Lhe lnLerpreLable SnÞs for an lndlvldual.
1he maln purpose of u1C geneLlc LesLlng ls empowermenL, allowlng cusLomers Lo obLaln wlde-ranglng
personal geneLlc lnformaLlon from a slngle source.

lndlvlduals may choose u1C geneLlc LesLlng for a varleLy of reasons, lncludlng:
• ldenLlfylng ancesLry, paLernlLy, or eLhnlclLy
• Seeklng dlsease-relaLed rlsk lnformaLlon Lo complemenL healLhcare and llfesLyle cholces
• lulfllllng personal curloslLy

1he u1C geneLlc LesLlng process ls qulck, affordable, and more accesslble Lhan lL would be Lo seek such
daLa from healLhcare provlders or oLher sources. Companles LhaL offer u1C geneLlc LesLlng are prlvaLely
held, and can provlde lndlvlduals wlLh healLh and ancesLry lnformaLlon aL cosLs ranglng from $99-$2000,
wlLh onllne daLa provlded wlLhln 6-8 weeks.

Well-known u1C geneLlc LesLlng companles lnclude:
• 23andMe, lnc.
• 8rlLalnsunA
• unAu1C
• lull Cenomes CorporaLlon
• Ceneux

A schemaLlc of a Lyplcal u1C geneLlc LesLlng servlce, as provlded by 23andMe, ls deplcLed ln 7+83'- 9.
AfLer orderlng, cusLomers are senL an aL-home sallva klL, whlch Lhey musL reglsLer, splL lnLo, and mall back
Lo Lhe company. Slnce sallva conLalns boLh whlLe blood cells and skln cells from Lhe lnner cheek, Lhls
provldes a means for Lhe company Lo access Lhe cusLomer's unA. AfLer recelvlng Lhe sallva sample, Lhe
company can begln laboraLory processlng Lowards SnÞ analysls of Lhe unA, whlch ls Lhe focus of Lhls
documenL. 1he raw and lnLerpreLaLlve resulLs are reporLed for Lhe cusLomer vla an lnformaLlonal webslLe
LhaL can lnclude dlscusslon boards and a means of connecLlng Lo relaLlves ldenLlfled vla Lhe LesLlng.

I|gure 2. 23andMe's u1C CeneLlc 1esLlng Servlce
(Source: 23andMe, hLLps://www.23andme.com)
*noLe: CLlA refers Lo Lhe luA's Cllnlcal LaboraLory lmprovemenL AmendmenLs

!"# !#%"&'%() *+,%#--
Pow does Lhe u1C geneLlc LesLlng company
esLabllsh healLh and ancesLry lnformaLlon
from Lhe cusLomer's sallva sample? 1hls ls
achleved vla an elaboraLe Lechnlcal process
LhaL occurs aL Lhe u1C geneLlc LesLlng
company slLe. LaboraLory Lechnlques and
compuLer analyses are employed.

As deplcLed by a schemaLlc ln 7+83'- 3, Lhe
Lechnlcal process lnvolves four maln sLeps,
some of whlch conLaln sub-sLeps. Cenerally,
Lhe process lncludes unA exLracLlon,
preparaLlon for hybrldlzaLlon vla
ampllflcaLlon and labellng, hybrldlzaLlon,
and analysls vla laser scannlng and

1. DNA Lxtract|on:

1he flrsL sLep ln Lhe geneLlc LesLlng process
ls Lo exLracL DNA (deoxyrlbonuclelc acld),
Lhe molecule LhaL conLalns all of an
lndlvldual's geneLlc lnformaLlon. 1he whlLe
blood cells and sklns cells found ln Lhe
cusLomer's sallva sample are sulLable for
Lhls exLracLlon. ln Lhls Lechnlque, unA ls
purlfled vla chemlcal and physlcal means
wlLhln varlous LesL Lubes. 1hls meLhod ls
essenLlal ln order Lo lsolaLe Lhe unA from
proLelns, llplds, and oLher molecules LhaL
are ln sallva, so LhaL lL may be furLher

I|gure 3. 1echnlcal Þrocess 8ehlnd
u1C CeneLlc 1esLlng

Sallva sample
1. unA LxLracLlon
2. ÞreparaLlon for

3. PybrldlzaLlon
4. Analysls
(Source: Wlklpedla, hLLp://wlklpedla.org/wlkl/)

As shown ln 7+83'- :, unA exLracLlon lLself lnvolves
varlous sLeps. lnlLlally, Lhe sample musL be comblned
wlLh cell lysls soluLlon (brand name lA8C) and
proLeln degradaLlon enzymes (brand name
ÞroLelnase k) Lo dlsrupL Lhe cell membranes and
break down Lhe proLelns LhaL make up Lhe cell,
respecLlvely. 1hls resulLs ln a heLerogeneous
soluLlon, whlch ls Lhen moved lnLo a speclal blndlng
LesL Lube for centr|fugat|on, Lhe use of a hlgh-power
splnnlng machlne Lo separaLe blologlcal molecules.
Cnce cenLrlfuged, unA remalns ln Lhe upper aqueous
layer, blndlng Lo an lnner porLlon of Lhe speclal LesL
Lube, whlle proLelns remaln ln Lhe lower phenol
layer, and are dlscarded (see 7+83'- ; for addlLlonal
lmagery). AfLerwards, Lhe unA ls cenLrlfuged
repeaLedly wlLh wash buffer (Lwo Llmes) and eluLlon
buffer (one Llme), Lo remove any lefLover reagenLs
and salLs from Lhe resulLlng phenol layers. ln Lhe
end, pure genomlc unA remalns.

2. Þreparat|on of the DNA for

1he nexL maln sLep ln Lhe process of u1C geneLlc LesLlng
ls Lo prepare Lhe exLracLed unA so LhaL lL ls ln an
explolLable form for hybrldlzaLlon. 1hls requlres
ampllflcaLlon of Lhe unA so LhaL Lhere ls enough Lo be
analyzed, followed by labellng of Lhe ampllfled unA so
LhaL lL can be monlLored durlng subsequenL analysls.

l. AmpllflcaLlon (ÞC8):
A blochemlcal process know as po|ymerase cha|n
react|on (ÞCk) ls employed ln order Lo
exponenLlally copy Lhe cusLomer's unA for
analysls (7+83'- <). 1he unA ls mlxed wlLh
enzymes, prlmers, and oLher sLablllzaLlon agenLs
ln a small LesL Lube. 1he enzymes serve Lo blnd Lo
Lhe unA molecule, and Lhe prlmers provlde a
place where blochemlcal ampllflcaLlon can begln.
I|gure S. ÞC8 AmpllflcaLlon
I|gure 4. unA LxLracLlon
(Source: Sclence CreaLlvlLy CuarLerly,
(Source: lavorgen 8loLech, hLLp://www.favorgen.com)


ÞC8 occurs wlLhln a Lhermal cycler machlne, whlch can lncrease and decrease LemperaLure
accordlng Lo a speclflc schedule. llrsL, Lhe sample ls heaLed, allowlng Lhe unA double-hellx Lo
denaLure lnLo Lwo separaLe sLrands. 1hen, Lhe sample ls cooled slowly, whlch allows Lhe enzymes
and prlmers Lo blnd Lo each sLrand. llnally, Lhe sample ls heaLed (lncubaLed) Lo an ldeal
LemperaLure for Lhe prlmers, whlch begln exLendlng each slngle sLrand lnLo new double sLrands.
AfLer each cycle, Lhe number of coples (compleLe double-hellces) of unA ls doubled. lor u1C
geneLlc LesLlng purposes, Lhe Lhermal cycler ls seL Lo compleLe around 30 cycles, resulLlng ln 2

coples of Lhe cusLomer's unA.

ll. Labellng (Coupllng):
AlLhough a sufflclenL amounL of unA ls produced by Lhe ÞC8 process, Lhls unA ls noL useful
unless lL ls labeled wlLh fluorescenL Lags LhaL can be monlLored durlng laLer sLeps (hybrldlzaLlon
and analysls). Labellng ls qulLe slmple, and requlres fluorescenL Lags ln soluLlon Lo be added
dlrecLly Lo Lhe LesL Lube conLalnlng Lhe ampllfled unA. 1yplcally, cyanlde dyes known as Cy3 and
Cy3 are added, slnce Lhey have green and red fluorescenL properLles, respecLlvely. Lach dye
blnds Lo a dlfferenL locaLlon on Lhe unA molecule, based on covalenL blndlng properLles. Slnce
Lhere are Lwo dyes, Lhe process of addlng Lhese dyes ls known as coup||ng.

3. nybr|d|zat|on of the Þrepared DNA to a M|croarray:

AfLer Lhe cusLomer's unA has been lsolaLed, ampllfled, and
labeled, Lhe acLual ºLesLlng" aspecL of geneLlc LesLlng can
begln. ln Lhls sLep, Lhe ldenLlflable SnÞs ln Lhe unA wlll be
recognlzed. 1he process, known as hybr|d|zat|on, refers Lo
placlng Lhe prepared unA on mlcroarray chlps, and Lhen
lncubaLlng Lo allow Lhe Lagged unA molecules Lo blnd
(hybrldlze) Lo Lhe chlps. 1hese hybrldlzed chlps can be
analyzed accordlng Lo Lhe blndlng paLLern LhaL occurs.

Speclflcally, u1C geneLlc LesLlng companles use mlcroarray
bead chlps LhaL are cusLomlzed for SnÞ genoLyplng, as
deplcLed ln 7+83'- =. Lach chlp ls essenLlally a small glass
sllde LhaL ls covered wlLh approxlmaLely 1 mllllon Llny lmmoblle beads, one for each SnÞ LhaL wlll be
LesLed. Lach bead has an aLLached probe, a chaln of small allele-speclflc ollgonucleoLldes (13-20
nucleoLlde base palrs). 1hese nucleoLlde probes are pleces of unA LhaL are complemenLary Lo Lhe slLes
on human unA LhaL represenL SnÞs. All probes are fluorescenLly labeled ln Lhe same way as Lhe
prepared unA. 1yplcally, u1C geneLlc LesLlng companles slmply purchase Lhese SnÞ mlcroarray bead
chlps from bloLechnology companles. 1he purchased bead chlps are deslgned Lo lnclude probes for SnÞs
wlLh assoclaLlons Lo healLh-relaLed LralLs, or SnÞs LhaL are ancesLral markers.
I|gure 6. SnÞ Mlcroarray Chlps
(Source: Sclence CreaLlve CuarLerly,

7+83'- 7 shows Lhe how Lhe sample unA
hybrldlzes Lo Lhe flxed probes on Lhe bead chlp.
llrsL, Lhe cusLomer's unA ls washed over Lhe
chlps and Lhe chlps are lncubaLed, allowlng for
hydrogen-bondlng Lo occur beLween Lhe sample
and Lhe probes. 1he sample unA wlll blnd
sLrongly Lo Lhe nucleoLldes on Lhe probe lf Lhe
probe ls fully complemenLary Lo lL. Any parLlally
complemenLary sLrands are weakly bonded and
are easlly washed off wlLh buffers, leavlng only
perfecLly complemenLary palrs aLLached.

4. Ana|ys|s of the nybr|d|zed

Cnce Lhe SnÞ mlcroarray bead chlps are sub[ecL Lo hybrldlzaLlon and washlng, Lhe chlps are analyzed ln
order Lo deLermlne whlch SnÞs are presenL. 1hls ls accompllshed vla laser scannlng and subsequenL
normallzaLlon by a compuLer program.

l. Laser Scannlng:
lf Lhe cusLomer's unA ls perfecLly
complemenLary Lo an SnÞ probe, Lhe
Cy3 and Cy3 fluorescenL Lags on Lhe
sample unA and Lhe probe wlll fluoresce ln an equlvalenL way,
lndlcaLlng LhaL Lhe cusLomer's genome lncludes LhaL speclflc
SnÞ (7+83'- >). 1he bead chlps are scanned by a laser sysLem,
whlch measures Lhe fluorescence as a llghL slgnal. uaLa
appears as a clusLer of green and red spoLs (7+83'- ?).

ll. normallzaLlon:
1he fluorescenL hybrldlzaLlon slgnals as provlded by Lhe
laser scanner musL be norma||zed, or processed as an
overall lmage, Lo quanLlfy whlch SnÞs are ln Lhe
cusLomer's genome. A hlgh-power compuLer program normallzes Lhe fluorescence daLa vla a
sLaLlsLlcal sysLem. 8ackground nolse ls removed, and Lhe sysLem accounLs for Lhe locaLlon of Lhe
slgnal on Lhe chlp whlle noLlng Lhe lnLenslLy of Lhe slgnal. 1hls normallzed daLa ls llnked Lo SnÞs
and Lhelr assoclaLed LralLs or markers, whlch compleLes Lhe Lechnlcal process of u1C geneLlc
LesLlng. llnally, Lhe daLa ls lnLerpreLed lnLo a form LhaL has healLh and ancesLral slgnlflcance.
1hese lnLerpreLaLlons are dlsplayed for Lhe cusLomer vla Lhe company's onllne webslLe.
I|gure 7. unA PybrldlzaLlon
I|gure 8. Laser Scannlng uaLa
(Source: Wlklpedla, hLLp://wlklpedla.org/wlkl/)
(Source: naLure, hLLp://www.naLure.com)

1he goal of u1C geneLlc LesLlng ls Lo empower lndlvlduals Lo obLaln rellable geneLlc lnformaLlon from a
slngle source. AfLer Lhe cusLomer's sallva sample ls recelved, Lechnlcal processlng employs laboraLory
and compuLer Lechnlques Lo obLaln, quanLlfy, and analyze daLa before lL ls lnLerpreLed as healLh and
ancesLry lnformaLlon for Lhe cusLomer. ln summary, Lhere are four maln sLeps Lo Lhe Lechnlcal
processlng, some of whlch conLaln sub-sLeps. unA ls exLracLed from Lhe sallva sample, ampllfled vla ÞC8,
and labeled wlLh fluorescenL Lags. 1he prepared unA ls sub[ecL Lo hybrldlzaLlon on an SnÞ mlcroarray
bead chlp, whlch ls quanLlfled vla laser scannlng and normallzed by a compuLer. 1he ensulng
lnLerpreLaLlon of Lhe daLa wlLh relaLlon Lo ancesLry and healLh rlsk ls Lhe mosL conLroverslal aspecL of Lhe
u1C geneLlc LesLlng servlce, buL lnLerpreLaLlon accuracy wlll llkely lmprove as Lhe relaLlonshlps beLween
SnÞs and phenoLype are furLher declphered. Cverall, u1C geneLlc LesLlng provldes a gllmpse aL whaL
bloLechnology ln geneLlcs can accompllsh, and ls of hlgh relevance Lo sLudenLs sLudylng Lhe llfe sclences.

!"#$% '()*+
ºPow lL works." @;)"4A-B 23andMe, lnc, n.d. Web. 24 March 2014.
!asmlne, larzana, eL al. "Whole-genome ampllflcaLlon enables accuraLe genoLyplng for mlcroarray-based
hlgh-denslLy slngle nucleoLlde polymorphlsm array." C)"0-' D1+4-(+&E&8F G+&()'H-'/ I
J'-,-"*+&" 17.12 (2008): 3499-3308.
Su, Þascal. "ulrecL-Lo-Consumer CeneLlc 1esLlng: A Comprehenslve vlew. "K5- L)E- M&3'")E &% N+&E&8F
)"4 (-4+0+"- 86.3 (2013): 339.
ºWhaL ls dlrecL-Lo-consumer geneLlc LesLlng?" O-"-*+0/ P&(- Q-%-'-"0-B u.S. naLlonal Llbrary of
Medlclne, 24 March 2014. Web. 24 March 2014. <hLLp://ghr.nlm.nlh.gov/

Image on t|t|e page.
unA double-hellx. ulglLal lmage. C'-)*+). n.p., n.d. Web. 24 March 2014.
I|gure 1.
SnÞs. ulglLal lmage. OE&//)'F. 8road lnsLlLuLe, 2014. Web. 24 March 2014.
I|gure 2. (cropped)
23andMe's u1C CeneLlc 1esLlng Servlce. ulglLal lmage. P&R +* S&'H/. 23andMe, LLC, 2007-2014. Web. 24
March 2014. <hLLps://www.23andme.com/howlLworks/>.
I|gure 3. (cropped, |abe|s ed|ted)
1echnlcal Þrocess 8ehlnd u1C CeneLlc 1esLlng. ulglLal lmage. .T! A+0'&)'')F DU1-'+(-"*. Wlklpedla, 3
March 2013. Web. 24 March 2014. <hLLp://en.wlklpedla.org/wlkl/unA_mlcroarray_experlmenL>.
I|gure 4.
unA LxLracLlon. ulglLal lmage. O-"&(+0 .T! DU*')0*+&" GE&&4 .T! A+"+ V A)U+ W+*. lavorgen 8loLech
CorporaLlon, 2009. Web. 22 March 2014.
I|gure S.
ÞC8 AmpllflcaLlon. ulglLal lmage. .T! 7+"8-'1'+"*+"8 +" *5- 2*)"4)'4+X)*+&" &% P-'N/ )"4 T3*')0-3*+0)E/.
Sclence CreaLlvlLy CuarLerly, 2014. Web. 22 March 2014.
I|gure 6.
SnÞ Mlcroarray Chlps. ulglLal lmage. K-/* L&3' .T! %&' .+/-)/-/ Y T& .&0*&' Q-Z3+'-4. 1lme Magazlne, 23
CcLober 2012. Web. 23 March 2014. <hLLp://healLhland.Llme.com/2012/10/23/drugsLore-genomes-

I|gure 7. (cropped, c|rc|es added)
unA PybrldlzaLlon. ulglLal lmage. .T! PFN'+4+X)*+&". Wlklpedla, 3 March 2013. Web. 24 March 2014.
I|gure 8. (cropped)
Laser Scannlng uaLa. ulglLal lmage. CF/*+0 %+N'&/+/ 0)''+-' /0'--"+"8[ \)E+4)*+&" &% ) "&,-E (-*5&4 3/+"8
G-)4C5+1 *-05"&E&8FB naLure, 13 May 2004. Web. 26 March 2014.