Abst ract
Submission: February 16, 2016
Accepted: September 6, 2016
Hum an DNA ext ract ion from w hole
saliva t hat was fresh or st ored for 3,
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prot ocols
Saliva when com pared t o blood collect ion has t he following advant ages: it requires no specialized personnel for collect ion, allows for rem ot e collect ion by t he pat ient , is painless, well accept ed by part icipant s, has decreased risks of disease t ransm ission, does not clot , can be frozen before DNA ext ract ion and possibly has a longer st orage t im e. Obj ect ive and Mat erial and Met hods: This st udy aim ed t o com pare t he quant it y and qualit y of hum an DNA ext ract ed from saliva t hat was fresh or frozen for t hree, six and t welve m ont hs using
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kit , prot ocol 2 – QI Aam p DNA m ini kit , prot ocol 3 – DNA ext ract ion using
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was analyzed using spect rophot om et ry, elect rophoresis and PCR. Result s: Result s indicat ed t hat t im e spent in st orage t ypically decreased t he DNA quant it y wit h t he except ion of prot ocol 1. The purit y of DNA was generally not affect ed by st orage t im es for t he com m ercial based prot ocols, w hile t he purit y of t he DNA sam ples ext ract ed by t he noncom m ercial prot ocols t ypically decreased w hen t he saliva was st ored longer. Only prot ocol 1 consist ent ly ext ract ed unfragm ent ed DNA sam ples. I n general, DNA sam ples ext ract ed
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t he prot ocol used, it was possible t o ext ract DNA in high quant it ies and of good qualit y using whole saliva, and furt herm ore, for t he purposes of DNA ext ract ion, saliva can be reliably st ored for relat ively long t im e periods. Conclusions: I n sum m ary, a com plicat ed pict ure em erges w hen t aking int o account t he ext ract ed DNA’s quant it y, purit y and qualit y; depending on a given researchers needs, one prot ocol’s part icular st rengt hs and cost s m ight be t he deciding fact or for it s em ploym ent .
Ke yw or ds: Saliva. DNA. Spect rophot om et ry. Elect rophoresis. Polym erase chain react ion.
Thais Francini GARBIERI1,2
Daniel Thomas BROZOSKI2
Thiago José DIONÍSIO2
Carlos Ferreira SANTOS²
Lucimara Teixeira das NEVES1,2
http://dx.doi.org/10.1590/1678-77572016-0046
1Universidade de São Paulo, Hospital de Reabilitação de Anomalias Craniofaciais, Bauru, SP, Brasil. 2Universidade de São Paulo, Faculdade de Odontologia de Bauru, Bauru, SP, Brasil.
148
J Appl Oral Sci.I nt roduct ion
For large scale genet ic st udies, t he am ount and
qualit y of DNA available from a sam ple is an essent ial
r equir em ent . Usually t he pr efer r ed sour ce for t he
collect ion of gen et ic m at er ial for t h ese st u dies is
peripheral blood1 because it yields large am ount s of '1$aWRJper m L of blood)16 t ypically free
of foreign DNA, yet t his procedure is not opt im um
since venipunct ure can be a painful experience and
has t he possibilit y of t ransm it t ing diseases4, requires
t rained personnel for collect ion, is com m only feared by
part icipant s causing volunt eers t o refuse t o part icipat e
in research14 and t he DNA in ext ract ed blood degrades
quick ly w it hout r efr igerat ion and m ust t y pically be
processed approxim at ely seven days aft er st orage16.
An ot h er im por t an t f act or t o con sider w h en u sin g
blood sam ples is t he presence of ferrous ions ( Fe2+)
t hat com pet e w it h Mg2+ ions, w hich can inhibit t he
polym erase chain react ion ( PCR) t echniques w idely
em ployed by m olecular st udies1. Addit ionally, t here
m ay be cult ural bar r ier s t o ex t ract ing blood11, and
except ional care m ost be t aken w hen sending blood
sam ples fr om differ ent locat ions. Last ly, analy zing
genom ic DNA from bone m arrow t ransplant recipient s
is unfeasible.
I n general, m olecular analy sis r equir es sev eral
processing st eps2 wit h DNA ext ract ion being one of t he
m ost im port ant st eps for t he success of a m olecular
genet ic st udy13. All t he reasons st at ed above have led
t o searches for alt ernat ive m et hods t o obt ain genet ic
m at erial for st udies requiring DNA, w it h saliva being
considered one of t he best candidat es13.
B r i e f l y , t h e s u b l i n g u a l , p a r o t i d a n d t h e
subm andibular glands secr et e saliva. Fur t her m or e, WKHVKHGGLQJRIWKHVXSHU¿FLDOOD\HURIHSLWKHOLDOFHOOV in h u m an or al m u cosa t h at occu r s appr ox im at ely
every 2.7 hours5 ult im at ely leads t o saliva com posed
of ~ 75% epit helial cells ( ~ 430,000 cells per m L5)
and ~ 25% leukocyt es ( 2 t o 136,000 cells per m L)7,19
depending on t he oral healt h of t he individual. Endler,
et al.7 ( 1999) found on average, at least 58% of t he
epit helial cells present in collect ed saliva sam ples t o
be viable w it h int act genom ic DNA7. How ever, t heir
ex t r act ion p r ot ocol, b ased on Sam b r ook , et al.1 7
( 1988) , t ypically ext ract ed t he m aj orit y of DNA from
leukocyt es and t hey hypot hesized t hat t heir prot ocol
m ore easily ext ract ed DNA from leukocyt es com pared
t o epit helial cells in t heir saliva sam ples from bone
m arrow t ransplant pat ient s17. How ever, it rem ains t o
be st udied if m odern DNA ext ract ion prot ocols differ
in t his respect from t he prot ocol based on Sam brook,
et al.17 ( 1988) .
Daw es ( 2003) , m oreover, found t hat saliva from
his volunt eers t ypically cont ained ~ 430,000 epit helial
cells per m L, and t hat , on average, each epit helial cell
had approxim at ely 80 t o 100 bact eria at t ached5. Thus
1 m L of hum an saliva cont ains a m ixt ure of DNA from
appr ox im at ely ~ 4.3x 105 epit helial cells, ~ 1.36x 105
leukocyt es and ~ 1.7x107 bact eria along wit h DNA from
ot her m icroorganism s found in t he oral cavit y. Besides
saliva, oral cells can be collect ed using a variet y of
m et hods, such as t he follow ing: swabs, cot t on spit
wads6, cy t ological br ushes, m out hwash w it h saline
and t reat ed Gut hrie cards9.
Ot h er com pon en t s of saliva su ch as en zy m es,
horm ones, im m unoglobulins and ot her biom olecules
can also int erfere w it h t he qualit y and quant it y of t he
genom ic DNA ext ract ed14. Overall, care should be used
for bot h DNA ext ract ion and t he preservat ion of t he
saliva14. I n part icular, care should be exercised w hen
ex am ining t he DNA fr om bone m ar r ow t ransplant
recipient s since saliva sam ples com m only produce a
chim eric m ixt ure of donor and recipient DNA. Endler,
et al.7 ( 1999) found chim eric DNA sam ples from 6
out of 8 saliva sam ples from bone m arrow t ransplant
recipient s w hereas Thiede, et al.19 ( 2000) found only
donor DNA in approxim at ely 10 t o 15% of recipient s
from saliva sam ples7,19.
Ordinarily, how ever, saliva can be a good source of
hum an DNA w hen com pared t o ot her alt ernat ive DNA
sources. Saliva can be easily collect ed by unt rained
individuals and ext ract ed DNA w it h a high m olecular
w eight can be st ored for long periods of t im e - up t o 5
years at room t em perat ure according t o DNA Genot ek
( D NA Gen o t ek ; Ot t a w a , On t a r i o, Ca n a d a )1 , 1 6 , 2 0.
Also, saliva collect ion is painless, w it h m inim al risk
of disease t r an sm ission . Sin ce saliva collect ion is
noninvasive, repeat ed collect ions are well t olerat ed by
m ost pat ient s15, and pat ient s can send saliva sam ples
by m ail, t hus, facilit at ing collect ion.
.FKOHU HW DO12 ( 2011) evaluat ed t he yield and
qualit y of genom ic DNA obt ained from fresh saliva and
saliva st ored for 4 and 8 days at room t em perat ure
u si n g o n e p r o t o co l1 2. Th ei r r esu l t s i n d i cat ed n o VLJQL¿FDQW GLIIHUHQFH EHWZHHQ WKH GLIIHUHQW VWRUDJH p er i o d s. Ho w ev er, t h er e ar e a f ew st u d i es t h at LQYHVWLJDWHGWKHSRVVLEOHLQÀXHQFHRIIUHH]LQJIRUORQJ
t erm st orage6, which is of fundam ent al im port ance for
t he creat ion of a saliva bank in large research cent ers.
Cu r r e n t l y, v a r i o u s D N A e x t r a c t i o n k i t s a r e FRPPHUFLDOO\ DYDLODEOH ZKLFK VWDQGDUGL]H HI¿FLHQW and convenient m et hods for obt aining genom ic DNA
from saliva14. Different ext ract ion m et hods can yield
d if f er en t am ou n t s of DNA w it h v ar y in g lev els of
purit y20. Oft en bot h t he quant it y and qualit y of DNA GLUHFWO\LQÀXHQFHWKHVXFFHVVDQGUHVXOWVRIVWXGLHV Know ing t he m axim um and ideal st orage durat ions
for saliva sam ples is valuable for m inim izing t he loss
of quant it y and/ or qualit y of DNA. Moreover, inferior
DNA ext ract ion can prevent t he successful com plet ion
of an experim ent al st udy, t herefore wast ing t im e and
m oney.
Thus, t his st udy aim s t o evaluat e t he quant it y and
qualit y of genom ic DNA obt ained from cells present in
fresh saliva and saliva frozen for t hree, six and t w elve PRQWKVXVLQJ¿YHGLIIHUHQW'1$H[WUDFWLRQSURWRFROV
Mat erial and m et hods
Sam ple collect ion
Tw ent y people w ere invit ed t o part icipat e in t his
st u dy. Eligibilit y cr it er ia in clu ded h ealt hy m ale or IHPDOHDGXOWVDJHG\HDUV$IWHUH[SODLQLQJWKH purpose of t he st udy and how t o part icipat e, a consent
form was com plet ed and signed by all part icipant s.
This st udy was appr oved by t he Et hics Com m it t ee
for Resear ch fr om t he Baur u School of Dent ist r y,
Un iv er sit y of São Pau lo, Bau r u , SP, Br azil, u n der
prot ocol num ber 192/ 2011.
%ULHÀ\SDUWLFLSDQWVH[SHFWRUDWHGDWOHDVWP/RI unst im ulat ed saliva int o a st erile, 50 m L polyet hylene
t ube at least 30 m inut es pr ior t o eat ing, dr inking,
sm oking or kissing t o m inim ize cont am inat es2. These
collect ion t ubes w ere m aint ained on ice and t he saliva
was aliquot ed int o st erile m icrocent rifuge t ubes ( 1.5
m L) . Next , each part icipant ’s aliquot ed sam ples w ere
st ored at - 20° C for t hree ( T3) , six ( T6) and t w elve
m ont hs ( T12) . The st orage periods st ipulat ed above for
t he whole saliva aim ed t o assess whet her t his freezing
w ould affect t he quant it y and qualit y of genom ic DNA
obt ained w hen com pared t o fresh saliva ( T0) .
DNA ext ract ion
7KH¿YHJHQRPLF'1$H[WUDFWLRQSURWRFROVXVHG ar e descr ibed below. For all com m er cial k it s, t h e PDQXIDFWXUHUV¶VSHFL¿FLQVWUXFWLRQVZHUHIROORZHG
Pr ot ocol 1 3URWRFRO XVLQJ PDQXDO SXUL¿FDWLRQ RI '1$ YLD WKH FRPPHUFLDO NLW 2UDJHQH '1$ Genot ek OG- 500; Ot t awa, Ont ario, Canada) . This kit
provides a collect ion t ube w it h 1 m L of suspension
buffer cont aining propriet ary reagent s t hat st abilize
t h e w h ole saliva sam ple pr ior t o DNA ex t r act ion . 6SHFL¿FDOO\DSSUR[LPDWHO\P/RIVDOLYDZDVPL[HG ZLWKWKHFROOHFWLRQEXIIHUDQG/RIWKLVPL[WXUH ZDV XVHG IRU WKH '1$ H[WUDFWLRQ 7KH ¿QDO HOXWLRQ YROXPHZDV/
Pr ot ocol 2 – Prot ocol using t he QI Aam p® DNA Mini
Kit ( Qiagen® 51306; Hilden, Nort h Rhine- West phalia,
Ger m any) . The DNA ext ract ion pr ot ocol uses silica
colum ns and no suspension buffer. Prot ocol 2 used /RIVDOLYDIRUWKH'1$H[WUDFWLRQDQGKDGD¿QDO HOXWLRQYROXPHRI/
Pr ot ocol 3 – Pr ot ocol for DNA ex t ract ion fr om
w hole saliva using am m onium acet at e, adapt ed from
Aidar an d Lin e1 2QH PRGL¿FDWLRQ WR WKLV
prot ocol included collect ing saliva wit hout a suspension
buffer1. Whole saliva w as cent r ifuged in a 1. 5 m L
m icr ocen t r if u ge t u be at 1 0 , 0 0 0 g f or 5 m in u t es.
The super nat ant was discar ded and t he pellet was
resuspended in 1 m L of ext ract ion buffer [ 10 m M Tris
– HCl; pH 7.8; 5 m M EDTA; 0.55% sodium dodecyl VXOIDWH6'6@1H[W/RISURWHLQDVH.PJ m L; Qiagen® 19133; Hilden, Nort h Rhine- West phalia,
Germ any) w ere added t o degrade prot eins. The t ubes
w ere vort exed and incubat ed in a wat er bat h at 56° C
overnight . Then sam ples w ere cent rifuged quickly ( t o FROOHFW DOO WKH OLTXLG WR WKH ERWWRP DQG / RI 10 M am m onium acet at e solut ion was added t o t he
t ubes, w hich w ere m ixed m anually for 3 t o 5 m inut es
follow ed by cent rifuging at 21,000 g for 15 m inut es at URRPWHPSHUDWXUH7KHQ/RIWKLVVXSHUQDWDQW ZDV WUDQVIHUUHG WR D QHZ WXEH DQG / RI FROG isopropyl alcohol was added followed by 15 seconds of
vort exing. The sam ples w ere placed in a refrigerat or
for 2 hours and t hen cent rifuged at 10,000 g for 20
m inut es at room t em perat ure. The supernat ant was
discarded w it h care t o not re- suspend t he pellet of
DNA; 1 m L of cold 70% et hanol was added t o t he
t ubes, w hich w ere t hen cent rifuged at 10,000 g for 5
m inut es. The supernat ant was again discarded, and
t he t ubes w ere left open for 4 t o 5 hours t o evaporat e
t he excess alcohol and t hen t he DNA was hydrat ed LQ/RIDXWRFODYHGGHLRQL]HGZDWHU/¿QDO elut ion volum e) .
150
J Appl Oral Sci. ZKROH VDOLYD ZLWK ,QVWD*HQH 0DWUL[ %LRUDG 7326030; Hercules, California, Unit ed St at es) . Tubescont aining 1.5 m L of w hole saliva sam ples w it hout
any suspension buffer w ere vort exed for uniform it y
of t he cont ent and cent r ifuged at 1 0 , 0 0 0 g for 5
m inut es at 4° C, t he supernat ant was discarded; 1 m L
of physiological saline was added t o t he t ubes w hich
w ere t hen vort exed unt il t he pellet was dissolved and
t hen vort exed for an addit ional 30 seconds. The t ubes
were cent rifuged at 10,000 g for 5 m inut es at 4° C, and
t he supernat ant was discarded. The sam e procedures
w it h t he addit ion of 1 m L of physiological saline w ere UHSHDWHG WZLFH PRUH 1H[W / RI ,QVWD*HQH Mat rix was added, t his m ixt ure was vort exed for 30
seconds and t he sam ples w ere incubat ed at 56° C for
30 m inut es. The t ubes w ere vort exed again for 10
seconds, and boiled at 100° C for 10 m inut es and again
vort exed for 10 seconds and cent rifuged at 15,000 g
for 5 m inut es at 4° C. Finally, t he supernat ant w it h t he
ext ract ed DNA was pipet t ed int o a new t ube leaving a ¿QDOHOXWLRQYROXPHRIDSSUR[LPDWHO\/
Pr ot ocol 5 – Pr ot ocol of DNA ex t ract ion fr om ZKROH VDOLYD ZLWK ,QVWD*HQH 0DWUL[ ,QVWD*HQH Mat rix dilut ed 1: 1) using Prot einase K and 1% SDS.
Tubes cont aining 1.5 m L of t ot al saliva w it hout any
suspension buffer w ere vort exed for uniform it y and
cent r ifuged at 10,000 g for 5 m inut es at 4° C, t he
supernat ant was discarded, and 1 m L of physiological
saline was added. The t ubes w ere vort exed unt il t he
precipit at e com plet ely dissolved and t hen vort exed for
an addit ional 30 seconds. The t ubes w ere cent rifuged
at 10,000 g for 5 m inut es at 4° C, and t he supernat ant
was discarded. The sam e procedures wit h t he addit ion
of 1 m L of physiological saline w ere repeat ed t w ice PRUH1H[W/RI'1$VHDQG51$VHIUHHZDWHUDQG /RI,QVWD*HQHZDVDGGHGWRWKHWXEHV w hich w ere t hen vort exed for 30 seconds. Sam ples
w ere incubat ed at 56° C for 30 m inut es and vort exed
for 10 seconds. The t ubes w ere boiled at 100° C for 10
m inut es and again vort exed for 10 seconds. Follow ing WKLVSURFHGXUH/RI3URWHLQDVH.JDQG /RI6'6ZHUHDGGHGWRWKHVDPSOHVDQGWKHQ vort exed and incubat ed at 65º C for 30 m inut es ( every
10 m inut es t he t ubes w ere m ixed) . Then, t he t ubes
were vort exed again and cent rifuged at 15,000 g for 5
m inut es at 4° C. Finally, t he supernat ant cont aining t he
ext ract ed DNA was pipet t ed int o a new t ube leaving a ¿QDOHOXWLRQYROXPHRIDSSUR[LPDWHO\/
Spect rophot om et ric analysis
A spect rophot om et er at wavelengt hs of 260 nm and QP1DQR'URS7KHUPR)LVKHU6FLHQW¿F Walt ham , Massachuset t s, Unit ed St at es) was used t o
quant ify and analyze t he condit ion of each ext ract ed
DNA sam ple fr om saliva. This equipm ent pr ov ides WZRLPSRUWDQWPHDVXUHVFRQFHQWUDWLRQQJ/ and ( 2) purit y ( via t he relat ive absorbance rat io of
260 nm / 280 nm ) for DNA w it h regards t o prot eins
and RNA. Sam ples w it h a rat io closer t o 1.8 indicat e
a relat ively pure DNA sam ple. The 260/ 280 nm rat ios
appreciably low er t han 1.6 are indicat ive of higher
prot ein cont am inat es. Sam ples w ere considered pure
if t he absorbance rat io was bet w een 1.6 and 2.0. This DQDO\VLVUHTXLUHV/RIHDFKVDPSOH
Elect rophoret ic analysis
To fur t her det er m ine t he qualit y and condit ion
of t he ex t ract ed DNA, sam ples of DNA fr om fr esh
saliva or saliva st ored for 3, 6 and 12 m ont hs using ¿YHGLIIHUHQWH[WUDFWLRQSURWRFROVIRUHDFKWLPHSRLQW w ere elect rophoresed using a 0.8% agarose gel ( 14
m m x 11.6 m m x ~ 5 m m ) in TAE buffer, Tris- acet at e
( 200 m M) w it h EDTA ( 50 m M) . Aft er t he DNA was
ex t ract ed, it w as st or ed for an addit ional 3 y ear s
b ef or e it w as an aly zed u sin g elect r op h or esis. I n
part icular, a st andard m olecular weight of 100 bp DNA
ladder ( I nvit rogen; Walt ham , Massachuset t s, Unit ed 6WDWHVDSRVLWLYHFRQWUROQJRI'1$/D QHJDWLYHFRQWURO/RIGG+22DQG/VDPSOHV IURPLQGLYLGXDOVZHUHORDGHGRQWRHDFKJHOZLWK/ of loading buffer ( NEOBI O product s for laborat ories,
cat alog num ber: NB- NT- 40501; Bot ucat u, SP, Brazil) .
The sam ples w ere loaded in t o t w o row s of w ells ( 2
m m x 1 m m x ~ 4 m m ) w it h 5 cm lanes and driven
by a pow er supply ( Loccus Biot echnologia LPS- 300V;
Cot ia, SP, Brazil) for ~ 150 m inut es using 40 m A, ~ 10
V per cm , at room t em perat ure. Aft er elect rophoresis,
t he DNA on t he gels was visualized using UV light
( Sigm a- Aldr ich T2 2 0 2 ; St . Louis, Missour i, Unit ed
St at es) , phot ographed using a digit al cam era ( Canon ,QF3&ŭWD7RN\R-DSDQDQGFDSWXUHGZLWK Doc- I t LS ( v er sion 6 . 0 . 0 ) sof t w ar e ( UVP; Up lan d ,
California, Unit ed St at es) .
PCR
Convent ional PCR was also used t o invest igat e t he
following t wo addit ional aspect s of t he ext ract ed DNA:
( 1) if any of t he prot ocols int roduced any variables
WKDW ZRXOG LQKLELW '1$ DPSOL¿FDWLRQ DQG XVLQJ SULPHUV ZLWK VSHFL¿FLW\ IRU KXPDQV WR YHULI\ LIKXPDQ'1$ZDVH[WUDFWHG%ULHÀ\3&5ZDVXVHG t o am plify exon 3 of t he int erferon regulat ory fact or
6 (I RF6) gene. The sequences of prim ers used w ere
5’-AGCTCTAGTAGATGGGAAAGGTG- 3’ ( sense st rained)
and 5’- CCAGAAAGGTCTGATGGTAGAAG- 3’ ( ant isense VWUDLQHG UHVXOWLQJ LQ DQ DPSOL¿HG IUDJPHQW RI bp. All t he reagent s used ( I nvit rogen PCR kit , cat alog
num ber 11615- 010; Walt ham , Massachuset t s, Unit ed
St at es) are out lined in Figure 1.
Co n v e n t i o n a l PCR w a s p e r f o r m e d u s i n g a
t herm ocycler ( Therm o Elect ron Corporat ion PxE0.5;
Walt h am , Massach u set t s, Un it ed St at es) w it h an
init ial denat uring st ep at 95° C for 4 m in, follow ed by F\FOHV RI DPSOL¿FDWLRQ (DFK DPSOL¿FDWLRQ F\FOH consist ed of denat urat ion for 30 seconds at 95° C,
annealing for 30 seconds at 63° C and an ext ension for
1 m inut e at 72° C. The sam ples w ere incubat ed for an
addit ional 7 m inut es at 72° C and m aint ained at 4º C
unt il t he t im e of rem oval.
7RFRQ¿UPDPSOL¿FDWLRQRIWKHIUDJPHQW/RI t he PCR product from each sam ple was m ixed w it h 2 /RIORDGLQJEXIIHU1(2%,2SURGXFWVIRUODERUDWRULHV cat alog num ber: NBNT40501; Bot ucat u, SP, Brazil) and
elect rophoresed in a 2% agarose gel using t he sam e
procedures out lined above.
St at ist ical analysis
Dat a w ere analyzed using Microsoft® Excel 2002
( v er si o n 1 0 . 6 8 7 1 . 6 8 7 0 ) , I BM® SPSS® st a t i st i cs YHUVLRQDQG*UDSK3DG3ULVP%ULHÀ\GDWD w ere t est ed for norm alit y using t he Shapiro- Wilk t est .
Non- norm ally dist ribut ed dat a w ere com pared using
t he Kruskal- Wallis t est . Mann- Whit ney U t est s w ere HPSOR\HGWRLGHQWLI\WKHVSHFL¿FGLIIHUHQFHVEHWZHHQ groups. Binary dat a (e.g. w het her DNA t est ed w it hin
purit y lim it s) w ere com pared using t he Pearson’s chi-VTXDUHGWHVW6WDWLVWLFDOVLJQL¿FDQFHZDVVHWDW Non- norm ally dist ribut ed dat a are represent ed by
box-and- whisker plot s report ing m edians wit h int erquart ile
ranges [ I QRs] .
Result s
DNA ext ract ion by each prot ocol was com pared
over each t im e t est ed and am ong each prot ocol using
– ( 1) spect rophot om et ry t o analyze bot h t he quant it y
yielded and t he relat ive purit y com pared t o RNA and
prot eins; ( 2) elect rophoresis t o analyze t he int egrit y
of t he DNA in t erm s of being int act or fragm ent ed;
and ( 3) convent ional PCR t o analyze whet her ext ract ed '1$FRXOGEHDPSOL¿HGXVLQJKXPDQVSHFL¿FSULPHUV These result s are sum m arized in Figure 2.
The ext ract ion of genom ic DNA from w hole saliva XVLQJ3URWRFROZDVVWDEOHDQGIDLUO\HI¿FLHQWDWHYHU\ t im e point t est ed. I t should be not ed t hat prot ocol 1
was t he only prot ocol w here collect ions w ere placed LQDVXVSHQVLRQEXIIHU:LWKD¿QDOHOXWLRQYROXPHRI /WKHDPRXQWRI'1$REWDLQHGE\WKLVSURWRFRO UDQJHGEHWZHHQJ>@DQGJ>@ ZLWK QR VLJQL¿FDQW GLIIHUHQFHV EHWZHHQ DQ\ RI WKH t est ed st orage t im e point s (p- value= 0.776,
Kruskal-Wallis t est , Figure 2Q t o 2T) . DNA ext ract ed from fresh VDOLYD7XVLQJSURWRFRO\LHOGHGVLJQL¿FDQWO\OHVV '1$ J >@ ZKHQ FRPSDUHG WR SURWRFROV DQG \LHOGLQJ DSSUR[LPDWHO\ J >@ SYDOXH DQG J >@ SYDOXH < 0.001) , r espect ively ( Figur e 2Q) . Wit h r espect t o
frozen sam ples, t he DNA yield obt ained wit h prot ocol 1
was sim ilar t o prot ocols 3 and 5 during t im e T3 ( saliva
st ored for 3 m ont hs) , and prot ocol 4 during T6 ( saliva
st ored for 6 m ont hs) and T12 ( saliva st ored for 12
Reagents Volume (μL) Final concentration
10x Buffer 5.0 1X
MgCl2 (50 mM) 1.5 1.5 mM
dNTPs (10 mM) 1.0 0.2 mM
Sense and antisense primers (25 μM) 0.8 0.4 μM each
Taq polymerase (5U / μL) 0.25 1.25 U
Dnase and RNAse free H2O 31.45
-DNA sample 10.0
-Total 50.0
-MgCl2: magnesium chloride
dNTPs: deoxynucleotides triphosphates
152
J
Appl Oral Sci.
Figure
2-Oragene™ kit; protocol 2 (2) used the QIAamp® DNA Mini kit; protocol 3 (3) used ammonium acetate, protocol 4 (4) used the InstaGene™ Matrix kit; protocol 5 (5) used the InstaGene™ kit with proteinase K and 1% SDS. Quantity in terms of concentration (ng/μL) was assessed using spectrophotometry and reported using medians and their respective interquartile ranges. (A) DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (B) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (C) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (D) DNA obtained by each of the 5 DNA extraction
extraction protocols; (K) Samples from saliva frozen for 6 months using each of the 5 DNA extraction protocols; (L) Samples from saliva frozen for 12 months using each of the 5 DNA extraction protocols. Conventional PCR using
frozen for 3 months; (O) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (P) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 12 months. Absolute quantity (μg) was assessed using spectrophotometry and reported using medians and their respective interquartile ranges; (Q) DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (R) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (S) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (T) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen
m ont hs) , as depict ed in Figures 2B t o 2D and 2R t o 2T.
W h e n c o m p a r e d t o p r o t o c o l 1 , p r o t o c o l 2
consist ent ly ext ract ed less DNA from frozen saliva at
all of t he invest igat ed t im e point s (p- value < 0.001,
Kruskal- Wallis t est , Figures 2B t o 2D and 2R t o 2T) UDQJLQJEHWZHHQJ>@DQGJ>@ Moreover, as report ed in Figures 2A t o 2D and 2Q t o
2T, prot ocol 2 recovered t he least am ount of DNA when
com pared t o all of t he ot her prot ocols at all t he t im e
point t est s w it h only t w o except ions, at fresh saliva DQG7$GGLWLRQDOO\DW7QJ/>@DQG7 QJ/>@'1$H[WUDFWLRQVXVLQJSURWRFROZHUH VLJQL¿FDQWO\ ORZHU ZKHQ FRPSDUHG WR WKH QJ/ [ 39] of DNA ext ract ed from fresh saliva using prot ocol
2 ( p- value < 0.002, Mann- Whit ney U t est ) .
I n general, t he st orage t im e of saliva affect ed t he
concent rat ion of DNA ext ract ed by prot ocol 3 ( Figure $WR'0RUHVSHFL¿FDOO\SURWRFROH[WUDFWHGWKH great est concent rat ion of DNA from fresh saliva ( 167 QJ/ >@ IROORZHG E\ 7 QJ/ >@ 7 QJ/>@DQG¿QDOO\7QJ/>@7KHVH FRQFHQWUDWLRQVRI'1$H[WUDFWHGDUHDOOVLJQL¿FDQWO\ different am ong each ot her w it h t he except ion of T3
versus T6 ( p- value= 0.074) . When DNA was ext ract ed
fr om fr esh saliva, t he t ot al am ount obt ained fr om SURWRFROV DQG ZHUH VLJQL¿FDQWO\ ORZHU WKDQ prot ocols 4 and 5 ( p- value < 0.0001, Kruskal- Wallis
t est , Figure 2Q) .
7KHVWRUDJHWLPHLQÀXHQFHGWKHDPRXQWRI'1$ ext ract ed by prot ocol 3, t he least am ount of DNA was
ext ract ed using prot ocol 3 from saliva st ored for 12 PRQWKVJ>@)LJXUH7ZKHQFRPSDUHG t o all ot her t im es and all ot her prot ocols.
Sig n if ican t d if f er en ces in t h e am ou n t of DNA
ext ract ed by prot ocol 4 w ere found am ong T0 ( 250 QJ/>@7QJ/>@DQGDWWKHWHVW WLPHVDIWHUPRQWKV7QJ/>@DQG7 QJ/>@DVGHSLFWHGLQ)LJXUHV$WR'SYDOXH < 0.05, Mann- Whit ney U t est ) .
Prot ocols 4 and 5 w ere able t o ext ract t he great est DPRXQWRI'1$IURPIUHVKVDOLYDJ>@ DQGJ>@UHVSHFWLYHO\LQD¿QDOHOXWLRQ YROXPHRIDSSUR[LPDWHO\/ZKHQFRPSDUHGWR all ot her prot ocols and t est ed t im e point s ( p- value
< 0.0001, Kruskal-Wallis t est , Figure 2Q) . Addit ionally, WKHUHZDVDVLJQL¿FDQWGLIIHUHQFHEHWZHHQWKHDPRXQW of DNA ext ract ed from fresh ( T0) versus frozen saliva
( T3, T6 and T12) as r epor t ed in Figur es 2A t o 2D
( p- value < 0.001, Mann- Whit ney U t est ) . Last ly, at
T12, prot ocol 5 yielded t he great est am ount of DNA J>@LQ/ZKHQFRPSDUHGWRSURWRFRO J>@LQ/DQGJ>@LQ /DVUHSRUWHGLQ)LJXUH7
I n t erm s of relat ive DNA purit y com pared t o RNA
an d p r ot ein s d et ect ed b y sp ect r op h ot om et r y, n o VLJQL¿FDQW GLIIHUHQFHV ZHUH GHWHFWHG LQ WKH SXULW\ of DNA ext ract ed from fresh saliva am ong prot ocols
1, 2, 3 and 4 ( p- value > 0.05, Mann- Whit ney U t est ,
Figures 2E t o 2H) . Conversely, prot ocol 5 did not have
any sam ples t hat t est ed w it hin t he 1.6 t o 2.0 spect ral
window from DNA ext ract ed from fresh saliva or saliva
t hat had been frozen for 6 or 12 m ont hs ( Figure 2E t o
2H) . DNA ext ract ed from saliva frozen for 3 m ont hs KDGVHWVRISURWRFROVWKDWZHUHVLJQL¿FDQWO\GLIIHUHQW SYDOXH)LJXUH)SURWRFROVDQGZHUH QRWVLJQL¿FDQWO\GLIIHUHQWSYDOXH ZLWKDERXW 84% of sam ples t est ing w it hin t he lim it s, w her eas SURWRFROVDQGZHUHQRWVLJQL¿FDQWO\GLIIHUHQW SYDOXH ZLWK OHVV WKDQ KDOI WKH VDPSOHV t est in g w it h in t h e pu r it y lim it s f or each of t h ese
prot ocols ( Figure 2F) . I n general, at all t he t est ed t im e
point s, prot ocols 1 and 2 had t he great est num ber
of sam ples w it hin t he purit y lim it s, w hile prot ocols
3 and 4 w ere variable. The percent age of sam ples
t hat w er e found t o be w it hin t he pur it y lim it s was
negat ively correlat ed w it h st orage t im e for prot ocol
4; conversely t he percent of pure DNA sam ples from
t he ot her ext ract ion prot ocols were not correlat ed wit h
st orage t im e. Prot ocol 5 rarely had sam ples t hat t est ed
posit ive for relat ive DNA purit y com pared t o RNA and
prot eins, i.e., t he rat io of t he absorbance at 260 nm
and 280 nm was alm ost always below 1.6.
Elect rophoret ic analysis wit h agarose gels was used
t o furt her charact erize t he condit ion of t he ext ract ed
DNA. Figure 3 provides a represent at ive exam ple of a
gel cont aining sam ples from t w o individuals at every
st orage t im e invest igat ed using each of t he prot ocols.
When com paring sam ples t aken from fresh DNA, aft er \HDUVRIVWRUDJH'1$H[WUDFWLRQVIURPIUHVKVDOLYD using prot ocol 1 yielded 100% unfragm ent ed DNA,
w hereas prot ocols 2, 3, 4 and 5 yielded 5% , 0% , 10%
and 20% , respect ively ( Figure 2I ) . I t was also found
t hat only prot ocol 1 yielded 100% or nearly 100% of
sam ples w it h unfragm ent ed DNA consist ent ly across
all t he ex am ined t im e point s ( Figur e 2I t o 2L and
Figure 3) . On t he ot her hand, prot ocols 2, 3, 4 and 5
rarely had unfragm ent ed DNA and, furt herm ore, as
154
J Appl Oral Sci.per cent age of DNA sam ples w er e undet ect able by
elect r ophor esis ( Figur e 2I t o 2L and Figur e 3) . I n
p ar t icu lar, t h e p er cen t ag e of sam p les f r om DNA
ext ract ions from saliva st ored for one year yielded t he
follow ing result s: prot ocol 1 had 95% unfragm ent ed
DNA and 5% fragm ent ed DNA; prot ocol 2 had 10%
unfragm ent ed DNA, 45% fragm ent ed DNA and 45%
undet ect able am ount s of DNA; prot ocol 3 had 32%
fragm ent ed DNA and 68% undet ect able am ount s of
DNA; prot ocol 4 had 59% fragm ent ed DNA and 41%
undet ect able am ount s of DNA; and prot ocol 5 had 38%
fragm ent ed DNA and 63% undet ect able am ount s of
DNA ( Figure 2L) .
Last ly, t h e ex t r act ed DNA w as an aly zed u sin g
convent ional PCR and elect rophoresis t o invest igat e WKH SHUFHQWDJH RI VDPSOHV WKDW FRXOG EH DPSOL¿HG E\ SULPHUV WKDW DUH VSHFL¿F IRU KXPDQ '1$ :KHQ using fresh saliva, t his analysis indicat ed t hat 95% of
t he sam ples under prot ocol 1, 100% under prot ocol
2, 90% under prot ocol 3, 80% under prot ocol 4 and
Figure 3- An example of a gel from 5 different extraction protocols when DNA was extracted from fresh saliva or saliva stored for 3, 6 or 12 months investigating whether samples were fragmented. DNA samples from 2 individuals (A, B) were electrophoresed using a 0.8% agarose gel in Tris-acetate (200 mM) with EDTA (50 mM) buffer. Lanes 1 and 25 contain the 100 bp molecular weight standard (M); lanes 2 and 26 contain the positive control (+), 115 ng of human DNA; lanes 3 and 27 contain the negative control (-), 8 μL of ddH2O; lanes 24 and 48 were left blank; all other lanes contain 8 μL of extracted DNA from either volunteer A or B. Protocol 1 (P1) used the Oragene™ kit; protocol 2 (P2) used the QIAamp® DNA Mini kit; protocol 3 (P3) used ammonium acetate, protocol 4 (P4) used the InstaGene™ Matrix kit;
protocol 5 (P5) used the InstaGene™ kit with proteinase K and 1% SDS
XQGHUSURWRFROFRXOGEHDPSOL¿HGZLWKKXPDQ VSHFL¿F SULPHUV )LJXUH 0 7KH VWRUDJH WLPH RI saliv a gen er ally did n ot af f ect t h e per cen t age of
sam ples posit ive for hum an DNA in all of t he prot ocols
t hat w ere t est ed ( Figure 2M t o 2P) . Only prot ocols 4 DQGKDGDVLJQL¿FDQWGLIIHUHQFHDPRQJWKHWHVWHG t im e point s, bot h differences w ere bet w een T6 and
T12 ( 70% versus 90% for prot ocol 4, respect ively;
p- value = 0.035 and 20% versus 0% for prot ocol 5,
respect ively; p- value = 0.035) . Overall, at nearly all
t he t im e point s exam ined, prot ocols 1 t hrough 4 w ere QRWVLJQL¿FDQWO\GLIIHUHQWIURPRQHDQRWKHUSYDOXH ! DQG WKHVH SURWRFROV ZHUH DOO VLJQL¿FDQWO\ great er t han prot ocol 5 ( p- value < 0.05) .
Discussion
When invest igat ing DNA, t he choice of t he prot ocol XVHGWRREWDLQJHQRPLF'1$FDQVLJQL¿FDQWO\LPSDFW WKH H[SHULPHQW $ VWXG\ PD\ UHTXLUH D VLPSOL¿HG collect ion syst em and t he long t erm st orage of sam ples ZKLOHPDLQWDLQLQJWKHDELOLW\WRH[WUDFWDVLJQL¿FDQW am ount of DNA t hat is r elat ively pur e. Depending
on t he cont ext , w hole saliva has several facet s t hat
m ake it an ideal candidat e for ext ract ing DNA. For
t his purpose, t his st udy invest igat ed t he quant it y and
qualit y of DNA ext ract ed from w hole saliva t hat was
fresh or frozen for 3, 6 or 12 m ont hs using 5 different
prot ocols. A com plicat ed pict ure em erged when t aking
int o account ( 1) t he quant it y of DNA ext ract ed, ( 2) t he
purit y of DNA com pared t o RNA and prot ein, ( 3) t he
condit ion of t he DNA ext ract ed w het her fragm ent ed RU XQIUDJPHQWHG DQG ¿QDOO\ LI HDFK SURWRFRO H[WUDFWHGVLJQL¿FDQWDPRXQWVRIKXPDQ'1$WKDWFRXOG EHDPSOL¿HGXVLQJFRQYHQWLRQDO3&5
I n general, m or e DNA could be ex t ract ed fr om
fresh saliva especially w hen using prot ocols 3, 4 and
5. Likew ise, fresh saliva generally allow ed for great er
percent ages of sam ples t hat w ere w it hin t he st andard
of relat ive DNA purit y com pared t o RNA and prot eins,
as det ect ed by spect r ophot om et r y. Longer st orage
t im es generally did not im pact t he DNA’s int egrit y,
and prot ocol 1 was m uch bet t er at ext ract ing DNA
t hat rem ained unfragm ent ed, w hereas prot ocols 2,
3, 4 and 5 ext ract ed DNA t hat was alm ost ent irely
fragm ent ed or undet ect able. I n general, t he st orage WLPHGLGQRWLQÀXHQFHWKHSHUFHQWDJHRI'1$VDPSOHV DPSOL¿HGE\KXPDQVSHFL¿FSULPHUVLQHDFKSURWRFRO Moreover, in every prot ocol except for prot ocol 5 m ost
'1$VDPSOHVZHUHDEOHWREHDPSOL¿HGXVLQJKXPDQ VSHFL¿FSULPHUV,WUHPDLQVWREHLQYHVWLJDWHGZKHWKHU procedures in prot ocol 5 eit her inhibit ed convent ional
PCR or if m ost of t he DNA ext ract ed was nonhum an. /DVWO\ LW VKRXOG EH QRWHG WKDW WKH ¿QDO HOXWLRQ volum es can be alt ered in all of t he prot ocols and t hat
( 2) in prot ocol 2 m ore DNA can be ext ract ed wit h m ore
elut ions w it h dim inishing concent rat ions. Therefore,
prot ocol 2 yields a varying am ount / concent rat ion of
DNA depending on t he num ber of elut ions used.
I t was expect ed t hat prot ocols 3, 4 and 5 w ould
yield m ore DNA since t hey ext ract ed DNA from 1.5
m L of saliva com pared t o prot ocols 1 and 2 w hich
used approxim at ely 0.2 m L. I t is unt est ed if m ult iple
collect ions by prot ocols 1 and 2 using t he sam e am ount
of st ar t ing m at er ial, 7.5 t im es t he am ount , w ould
t ruly yield 7.5 t im es t he am ount t hat was collect ed.
How ever, if t his assum pt ion is valid, t hen pr ot ocol ZRXOG EH PRUH HI¿FLHQW DW H[WUDFWLQJ '1$ IURP saliva w hen com pared t o prot ocols 2 and 3 and m ore
com parable t o prot ocols 4 and 5. That is, prot ocol 1 ZRXOGKDYH\LHOGHGDSSUR[LPDWHO\JRI'1$ IURPIUHVKVDOLYDJRI'1$IURPVDOLYDIUR]HQ IRUPRQWKVJRI'1$IURPVDOLYDIUR]HQIRU PRQWKVDQGJRI'1$IURPVDOLYDIUR]HQIRU 12 m ont hs; w hereas prot ocol 2 w ould have yielded JRI'1$IURPIUHVKVDOLYDJRI'1$IURP VDOLYDIUR]HQIRUPRQWKVJRI'1$IURPVDOLYD IUR]HQIRUPRQWKVDQGJRI'1$IURPVDOLYD frozen for 12 m ont hs. How ever, m ult iple collect ions
from prot ocols 1 and 2 would furt her increase t he cost
and ext ract ion t im e. I t should be st ressed t hat each
prot ocol was being evaluat ed alone and t hat all of t he
facet s t est ed ( quant it y, purit y, int egrit y and abilit y t o EH DPSOL¿HG E\ KXPDQ VSHFL¿F SULPHUV VKRXOG EH evaluat ed w hen choosing a prot ocol.
Prot ocol 1 was t he only prot ocol w here saliva was
collect ed and placed int o a suspension buffer, and it
was also t he only prot ocol w here t he st orage t im e GLGQRWVLJQL¿FDQWO\DIIHFWWKHFRQFHQWUDWLRQDPRXQW of DNA ext ract ed. Addit ionally, at every t im e point
t est ed, t he sam ples of DNA ext ract ed using prot ocol
1 had t he great est percent ages of purit y, t he m ost
unfragm ent ed sam ples and nearly all sam ples t est ed \LHOGHG SRVLWLYH UHVXOWV ZKHQ XVLQJ KXPDQ VSHFL¿F prim ers. Perhaps t he addit ion of a suspension buffer
in prot ocol 1 was inst rum ent al in preserving t he saliva
for DNA ext ract ion and keeping t he DNA unfragm ent ed
156
J Appl Oral Sci.Pr ot ocol 2 r ecov er ed t he least am ount of DNA
w hen com pared t o nearly all of t he ot her prot ocols
at all t h e t im e poin t s t est ed. Alt h ou gh , as n ot ed
abov e, addit ional elut ions m ay y ield m or e ov erall
DNA from t he spin colum n, but t his also reduces t he
concent rat ion. Sim ilarly t o prot ocol 1, t he percent age
of sam ples wit hin t he purit y t hreshold were unaffect ed
by st orage t im e and m ost sam ples w ere w it hin t he
accept ed t hreshold for purit y. However, only a few DNA
sam ples, at various t im e point s, w ere unfragm ent ed.
As dem onst rat ed by t he PCR result s in t his st udy, it is
st ill possible t o invest igat e different param et ers w it h
fragm ent ed DNA, but caut ion should be exer cised
w hen using prot ocol 2 for som e genet ic experim ent s.
DNA ex t r act ion u sin g pr ot ocol 3 w as sen sit iv e
bot h t o saliva being frozen and t o being st ored for ORQJHUSHULRGVRIWLPH0RUHVSHFL¿FDOO\SURWRFRO ex t ract ed t he gr eat est concent rat ion of DNA fr om IUHVK VDOLYD ZKLFK ZDV VLJQL¿FDQWO\ JUHDWHU ZKHQ com pared t o saliva t hat had been frozen for 3, 6 and
12 m ont hs. Com pared t o t he ot her prot ocols, prot ocol
3 ex t ract ed t he least am ount of DNA w hen saliva
w as st or ed for 12 m ont hs, w hile DNA ex t ract ions
from fresh saliva w ere great er t han prot ocols 1 and
2 ( unnorm alized for t he st art ing am ount of saliva) ,
but less t han prot ocols 4 and 5. The percent age of
sam ples t est ing w it hin t he purit y lim it s was variable
am ong t he st orage t im es invest igat ed w hen using
prot ocol 3. I n t erm s of int egrit y, sim ilarly t o prot ocols
2 , 3 an d 5 , DNA ex t r act ed u sin g pr ot ocol 3 w as
com plet ely fragm ent ed at all of t he t im e point s t hat
w ere invest igat ed w it h t he except ion of T6 w here 6%
of t he sam ples w ere unfragm ent ed. Last ly, alm ost all
DNA sam ples ext ract ed using prot ocol 3 at every t im e
point exam ined t est ed posit ive for hum an DNA.
Pr ot ocol 4 t ypically yielded t he second gr eat est
am ount of DNA from saliva sam ples, and, if st rict ly
look ing at hum an DNA, t he m ost am ount of DNA
w hen com par ed t o t he ot her pr ot ocols. The pur it y
of DNA ex t r act ed u sin g pr ot ocol 4 w as adv er sely
affect ed by st orage t im e, and t he percent age of DNA
sam ples w it hin t he accept able purit y t hreshold w ere
negat ively correlat ed wit h st orage t im e. Prot ocol 4 did
ext ract unfragm ent ed DNA in a few sam ples, sim ilar t o
prot ocols 2, 3 and 5, but not nearly as well as prot ocol
1. Alm ost all DNA sam ples ext ract ed using prot ocol
4, at every t im e point exam ined t est ed posit ive for
hum an DNA.
Al t h o u g h p r o t o co l 5 y i e l d e d t h e m o st D N A
f r om f r esh saliv a, n ear ly all of t h e DNA sam ples
ex t ract ed u sin g t h is pr ot ocol w er e n ot w it h in t h e
accept able relat ive DNA lim it s for purit y as assessed
by spect r ophot om et r y. Many of t he sam ples m ust
have cont ained m uch great er am ount s of RNA and/
or prot eins. I ndeed, rarely w ere DNA sam ples from
prot ocol 5 found t o be unfragm ent ed or even det ect able
using elect rophoret ic analysis, and furt herm ore, m ost VDPSOHV ZHUH QRW DPSOL¿HG E\ 3&5 XVLQJ KXPDQ VSHFL¿FSULPHUV)RUWKDWPDWWHULWUHPDLQVXQNQRZQ precisely how m uch any prot ocol m ay direct ly int erfere
w it h convent ional PCR com pared t o j ust sim ply not H[WUDFWLQJHQRXJKKXPDQ'1$ZLWKVXI¿FLHQWTXDQWLW\ and/ or int egrit y.
As in t he present st udy, t here are several ot her
st udies t hat aim ed t o est ablish w hich DNA ext ract ion IURPVDOLYDSURWRFROVZHUHWKHPRVWHI¿FLHQW1,9,12,18,20.
Com m er cial k it s ar e generally t he m ost com m only
used, generally present ing m ore consist ent result s.
This consist ency of DNA ext ract ion from saliva using
t he QI Aam p DNA kit is apparent w hen com paring t he
result s of t his st udy w it h ot her invest igat ions18,20. The
sam e can be said w hen com paring t his st udy’s result s
t o ot her invest igat ions w hen looking at t he result s
obt ain ed by u sin g t h e Or agen e Gen ot ek k it s2 0. I t
should be not ed t hat m any of t hese ot her st udies were
perform ed w it h saliva t hat was not st ored below 0° C. %ULHÀ\DJURXSRIUHVHDUFKHUV18 st ored saliva at
- 20° C for 6 m ont hs using t he QI Aam p DNA kit for DNA
ext ract ion. When com par ing t he concent rat ion and
qualit y of DNA ext ract ed from t his saliva t o t he present
st udy, at t he sam e st orage durat ions, t he result s are
in close agr eem en t . Fu r t h er m or e, t h e fr eezin g of
saliva for 11 years using t he sam e condit ions6 w it h
t he QI Aam p DNA kit also had result s sim ilar t o t hose
of t he present st udy w it h saliva frozen for 12 m ont hs.
Fur t her m or e, t he Oragene DNA k it ( DNA Genot ek )
st at es t hat viable DNA can be ext ract ed reliably from VDOLYD VWRUHG LQGH¿QLWHO\ DW WHPSHUDWXUHV EHWZHHQ - 1 5 ° C an d - 2 0 ° C an d st or ed for sev er al y ear s at
room t em perat ures. This prot ocol produced consist ent
result s in t erm s of quant it y and qualit y at t he t im e
point s invest igat ed in t his st udy and w hen com pared
t o ot her st udies in t he lit erat ure. I n part icular, t he
result s from t his st udy and a st udy by Ng, et al.14 KDGD'1$FRQFHQWUDWLRQRIDERXWQJ/ w it h m ost sam ples being w it hin t he purit y t hreshold
based on spect rophot om et ry.
The com m ercial kit s m ent ioned above are easy t o
handle and very consist ent , but som et im es unviable
due t o cost s. Wit h t his in m ind, som e st udies1 , 1 2,
including t he present one, t est ed low- cost alt ernat ive
prot ocols such as t he prot ocols t hat used am m onium
acet at e or t he m at rix I nst agene Bio- Rad reagent . Wit h
respect t o t he prot ocol using am m onium acet at e, ot her
researchers invest igat ed DNA ext ract ion from saliva
st ored at room t em perat ure for 1, 2, 4, 8, 15 and
30 days1,12. These result s w ere sim ilar t o w hat was
found in t his st udy regarding bot h t he concent rat ion
and qualit y of DNA. To dat e, no st udies w ere found
t h at in v est ig at ed DNA ex t r act ion p r ot ocols u sin g
am m onium acet at e w here DNA was ext ract ed from
saliva t hat was frozen. Not ably, saliva frozen for 6 DQGPRQWKVLQWKLVVWXG\GLGQRWHI¿FLHQWO\\LHOG DNA w hen using t he am m onium acet at e pr ot ocol,
and t hus, st udies hoping t o ex t ract int act DNA in VXI¿FLHQWTXDQWLWLHVIURPIUR]HQVDOLYDVKRXOGQRWXVH t his prot ocol. The ot her low- cost prot ocol using t he
I nst agene Mat rix from Bio- Rad had int riguing result s,
ext ract ing large am ount s of DNA but wit h less qualit y/
purit y w hen com pared t o som e of t he ot her prot ocols
used. However, as t im e st orage of t he saliva increased
bot h t he quant it y and qualit y of t he DNA ext ract ed
dim inished.
A st u d y b y Go o d e, et al .8 ( 2 0 1 4 ) d et ai l s an
opt im ized procedure for ext ract ing DNA using reagent s
from t he Puregene ext ract ion kit ( Qiagen)8. They found
t hat using a reagent volum e sm aller t hen recom m end
by t he m anufact urer did not com prom ise t he am ount
of DNA ext ract ed and opt im ized cost s. Not ably, t heir
prot ocol is sim ilar t o prot ocol 3 used in t his st udy.
Furt her exam inat ion m ay reveal t hat cost s and DNA
ex t r act ion s can be f u r t h er opt im ized in a sim ilar
m anner.
A sear ch of t h e lit er at u r e r ev ealed on ly on e
DNA ext ract ion from m icroorganism s from cult ured
m edium s using t he I nst agene m at rix prot ocol3,10. This
st udy not ed t he successful ext ract ion of DNA from t his VRXUFHEXWLWUHPDLQVGLI¿FXOWWRFRPSDUHWKLVUHVXOW w it h t he present st udy w here t he ext ract ions w ere
from w hole saliva.
Conclusion
When v iew ed w it h t he per spect ive gained fr om
t h is st u d y an d f r om ot h er in d ep en d en t st u d ies,
it is est im at ed t h at com m er cial k it s, som et im es
independent of t he st orage t im e, provide consist ent
result s in t erm s of t he concent rat ion and purit y of DNA
ext ract ed from w hole saliva, especially w hen dealing
w it h saliva t hat has been frozen and/ or st ored for a
long t im e. I f w hole genom ic DNA is needed, t hen only
prot ocol 1 can be recom m ended. The less expensive ODERUDWRU\ SURWRFROV SURYLGHG OHVV VXI¿FLHQW UHVXOWV w it h st or ed fr ozen saliva; how ever, fr esh saliva or
saliva st ored for short durat ions m ight be adequat e IRUREWDLQLQJ'1$ZLWKDVXI¿FLHQWTXDQWLW\DQGSXULW\ albeit fragm ent ed. Alt hough, as st r essed above, a
com plicat ed pict ure em erges when t aking int o account
t he ext ract ed DNA’s quant it y, purit y, qualit y and t he
prot ocols abilit y t o provide decent st art ing m at erial for
PCR, and, depending on a given researchers needs,
one prot ocol’s part icular st rengt hs and cost s m ight be
t he deciding fact or for it s em ploym ent .
Acknow ledgem ent s
7KLVVWXG\ZDV¿QDQFHGE\WKH6mR3DXOR5HVHDUFK Foundat ion ( FAPESP 2011/ 22434- 7 and 2009/
53848-1) . We also w ould like t o t hank Dr. Marilanda Ferreira
Bellini for her helpful cont ribut ions t o t his m anuscript .
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