Department of Chemical & Biomolecular

Engineering
THE NATIONAL
UNIVERIT! of
IN"A#ORE
Chemical Engineering Process Laboratory I
CN2108
Experiment B3:
Thermodynamics and Kinetics of DNA Hybridization
Name (Matric No.): Chen !eling Cheryl ("0#$%#8r)
Chie& 'ian(ao )eli* ("0#$+#%h)
Ch!a ,he -ei ("0#$+8.&)

/ro!0 : M2
1ate o2 : $343200$
E*0eriment
1emonstrator5s :
6ignat!re /781E:
CONTENT A!E
"#mmary$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$3
%& 'ntrod#ction$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$(
)& Theoretica* Bac+,ro#nd$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$-
2.1 1N8
2.2 8n intro9!ction on the 7eal time PC7
2.4 :he Nee9 2or 7eal;:ime PC7
2.# 7eal;:ime PC7 Chemistries
2.. <!anti2ication
2.$ 7eal;:ime PC7 1ata 8nalysis
2.+ Non;PC7 800lications
3& Experimenta*$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$%.
4.1 800arat!s
4.2 E*0erimental Proce9!re
4.2.1. 1il!tion o2 each o2 the # single stran9e9 1N8s ($4= $#= $.= $$)
4.2.2 Pre0aration o2 testing sam0les in sam0le >ials (1..ml)
4.2.2.1 Matche9 9o!ble stran9e9 1N8 $43$#
4.2.2.2 Mismatche9 9o!ble stran9e9 1N8 $43$.
4.2.2.4 Mismatche9 9o!ble stran9e9 1N8 $43$$
4.2.2.# Pre0are 4 i9entical ?lan@ 6am0les
4.2.2.. 8nalyAe sam0les !sing 7eal;:ime PC7 system
(& /es#*ts$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$%0
#.1 /ra0h 2or (ybri9 1N8 $4 &ith $# (sam0le 8124= (igh Conc)
#.2 /ra0h 2or (ybri9 1N8 $4 &ith $# (sam0le ?124= Lo& Conc)
#.4 /ra0h 2or (ybri9 1N8 $4 &ith $. (sam0le C124= (igh Conc)
#.# /ra0h 2or (ybri9 1N8 $4 &ith $. (sam0le 1124= Lo& Conc)
#.. /ra0h 2or (ybri9 1N8 $4 &ith $$(sam0le E124= (igh Conc)
#.$ /ra0h 2or (ybri9 1N8 $4 &ith $$(sam0le )124= Lo& Conc)
#.+ 6am0le ?lan@124
.& Disc#ssion$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$)-
-& Conc*#sion$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$3%
1& /eferences$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$33
2& Notation$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$33
Appendix A 3 /es#*ts tab*es
2
"#mmary
-e are !sing the real time PC7 to in>estigate the e22ect o2 com0lementary
seB!encing on 1N8 stability. :his is 9one !sing 1N8;$4= 1N8;$#= 1N8;$. an9
1N8;$$ at 9i22erent concentrations !sing the 0henomena that 6C?7 green
2l!orescence 9ye intercalates &ith 9o!ble stran9e9 1N8. be seen that mismatch
o2 1N8 seB!ences has an e22ect on the hybri9iAation39ehybri9iAation 0rocess=
&hereby the greater the 9egree o2 mismatch the lo&er the melting 0oint o2 the
hybri9. 8lso= mismatch at the centre o2 the hybri9 ten9s to ha>e a more
0rono!nce9 e22ect on the 0rocess as com0are9 to mismatch at the en9s.
8ll these can be seen 2rom the gra0h o2 -d(RFU)/dT >ers!s tem0erat!re &hereby
mismatch at the center ten9 to ha>e the lo&est tem0erat!re.
3
%&'ntrod#ction
-hen a 9o!ble stran9e9 1N8 (or 9o!ble heli* 1N8) is heate9 to 9enat!ration
tem0erat!re= the 9o!ble heli* &ill se0arate or DmeltE to 2orm t&o single stran9s.
:he coole9 9o!ble helices &ill re2orm (renat!ration) at regions o2 seB!ence
com0limentarity. :his techniB!e is !se2!l in 9etermining the seB!ence similarity in
1N8s o2 9i22erent origin an9 the amo!nt o2 seB!ence re0etition &ithin one stran9
o2 1N8.
(ybri9iAation again 9e0en9s on the basic 0rinci0le o2 the n!mber o2 hy9rogen
bon9ing bet&een /C an9 8: bon9s in n!cleic aci9s. :he 0rinci0les in the
hybri9iAation ste0s are as 2ollo&:
1. 1N8 stran9s are se0arate9 by brea@ing the hy9rogen bon9s bet&een the
bases &ith heat to 0ro9!ce single stran9s.
2. 6tran9 se0aration is strictly 9e0en9ent !0on t&o 2actors: :he tem0erat!re
(amo!nt o2 heat energy) an9 the n!mber o2 hy9rogen bon9s (an incorrect
base 0airing 9oes not 2orm ( bon9s)
4. -hen the tem0erat!re 9ro0s= single stran9e9 1N8 in the same sol!tion
that are com0lementary s0ontaneo!sly come together by 0airing !0
thro!gh their res0ecti>e 8: an9 /C associations. :he strength o2 their
4
s!bseB!ent association 9e0en9s on the tem0erat!re an9 the total n!mber
o2 matching base 0airs.
Fne analytical techniB!e= the Polymerase Chain 7eaction (PC7)= has been
2o!n9 to be e*tremely !se2!l in 9etermining seB!ence similarity among 1N8s o2
9i22erent origins= as &ell as the amo!nt o2 seB!ence re0etition &ithin one 1N8.
:he a9>ent o2 7eal;:ime PC7 has allo&e9 2or more &i9es0rea9 !se o2 PC7
assays 2or 9iagnostic 0!r0oses. In this e*0eriment= 7eal;:ime PC7 B!anti2ication
techniB!es &ill be em0loye9 to monitor the 2ormation o2 9o!ble;stran9e9 1N8 at
9i22erent tem0erat!res so as to !n9erstan9 &hen hybri9iAation an9
9ehybri9isation occ!rs an9 ho& they are a22ecte9 by a seB!ence mismatch.
PC7= ho&e>er= &ill not be carrie9 o!t.
Ob4ecti5e:
1. /ain an !n9erstan9ing o2 1N8 hybri9iAation G the 2ormation o2 a 9o!ble
stran9e9 1N8 oligon!cleoti9e molec!le 2rom t&o single stran9e9
molec!les.
2. "n9erstan9 the critical im0ortance o2 seB!ence com0lementary in 1N8
hybri9iAation.
5
)& Theoretica* Bac+,ro#nd
2.1 DNA
1eo*yribon!cleic aci9 (1N8) is the genetic material o2 all cells= carrying
in2ormation in the 2orm o2 a genetic co9e 2rom cell to cell an9 2rom 0arent to
o22s0ring. 8 gene is a segment o2 1N8 that 9etermines the seB!ence o2
nitrogeno!s bases in ribon!cleic aci9 (7N8)= &hich in t!rn 9etermines the
seB!ence o2 amino aci9s in a s0eci2ic 0rotein. :he characteristics o2 an organism
are 9etermine9 by the gene(s) an9 the res!lting 0rotein(s) that it is able to
synthesiAe. :he 0roteins 0ro>i9e str!ct!re an9 catalyAe chemical reactions=
among other things. :his constit!tes the central 9ogma o2 mo9ern biology= as
s!mmariAe9 belo&:
1N8 1N8
Replication
1N8 7N8 P7F:EIN
:ranscri0tion :ranslation
6
Co! can see that the 2lo& o2 genetic in2ormation 2rom 1N8 in>ol>es three main
ste0s:
(1) re0lication o2 the genetic in2ormation in 1N8= &hich occ!rs in the synthesis
0hase o2 inter0hase in the cell cycle= 0rior to cell 9i>ision=
(2) transcri0tion o2 1N8 into 7N8s=
(4) translation o2 m7N8 into 0roteins.
1N8 an9 7N8 are com0ose9 o2 chemical s!b!nits @no&n as n!cleoti9es. Each
n!cleoti9e is ma9e !0 o2 a 0hos0hate gro!0= a 0entose s!gar= an9 one o2 the
2o!r nitrogeno!s bases. 1N8 has 2o!r nitrogeno!s bases: a9enine= g!anine=
cytosine an9 thymine. 7N8 also is com0ose9 o2 2o!r bases: a9enine= g!anine=
cytosine an9 !racil. :he st!9ent sho!l9 remember that a9enine an9 g!anine are
H9o!ble ringH com0o!n9s categoriAe9 as 0!rines= &hereas cytosine= thymine an9
!racil are Hsingle ringH com0o!n9s categoriAe9 as 0yrimi9ines.
1eo*yribon!cleoti9es contain the s!gar 9eo*yribose. 7ibon!cleoti9es= &hich are
the b!il9ing bloc@s 2or 7N8 synthesis= contain the s!gar ribose. :he n!cleoti9es
are Ioine9 together by a single ty0e o2 0hos0ho9iester lin@age to 2orm long chains
(stran9s) o2 1N8= &hich res!lts in the 2ormation o2 t&o stran9s arrange9 in a
9o!ble helical 2orm. In 1N8= a9enine (8) is al&ays hy9rogen;bon9e9 to thymine
7
(:) an9 g!anine (/) is al&ays hy9rogen;bon9e9 to cytosine (C). 6ince the t&o
stran9s o2 the 1N8 base;0air &ith each other= they are sai9 to be com0lementary
stran9s. :his is @no&n as the r!le o2 base 0airing.
2.2 An introduction on the Real time PCR
:he M' Mini gra9ient cycler o22ers:
8 thermal gra9ient 2eat!re that allo&s sim!ltaneo!s inc!bation at 8
9i22erent tem0erat!res so yo! can o0timiAe reactions 2or ma*im!m
e22iciency an9 acc!rate B!antitation
7emar@ably ra0i9 arri>al at thermal !ni2ormity= &hich 0ro9!ces the
0recision nee9e9 2or sensiti>e assays s!ch as B!antitati>e PC7
8 Peltier heat 0!m0= b!ilt in;ho!se= that 0ro9!ces B!ic@ ram0ing an9
acc!rate tem0erat!res to ens!re 2ast re0ro9!cible r!ns
8 #8;&ell sam0le bloc@ ca00e9 &ith an a9I!stable heate9 li9 that
accommo9ates both 2!ll;height an9 lo&;0ro2ile t!bes an9 0lates= so yo!
can r!n lo&;>ol!me reactions &ith minimal sam0le loss
:e*t an9 gra0hical 9is0lay o0tions= &ith gra0hical e9iting 2or ra0i9ly
mo9i2ying 0rograms
Light &eight an9 com0act siAe that allo& the instr!ment to 2it I!st abo!t
any&here
8
2.3 The Need for Real-Time PCR
M!ch o2 the technical e22ort in>ol>e9 in stan9ar9 PC7 is no& 9irecte9 to&ar9
0ositi>e recognition o2 the am0licons. :he im0ortant metho9s o2 0ost;PC7
analysis rely on either the siAe or seB!ence o2 the am0licon. /el electro0horesis
is o2ten !se9 to meas!re the siAe o2 the am0licon an9 this is both ine*0ensi>e
an9 sim0le to im0lement. "n2ort!nately= siAe analysis has limite9 s0eci2icity since
9i22erent molec!les o2 a00ro*imately the same molec!lar &eight cannot be
9isting!ishe9. ConseB!ently= gel electro0horesis alone is not a s!22icient PC7
en9;0oint in many instances= incl!9ing most clinical a00lications.
Characterisation o2 the 0ro9!ct by its seB!ence is 2ar more reliable an9
in2ormati>e. Probe hybri9isation assays 2or this 0!r0ose are a>ailable b!t many
are m!lti;ste0 0roce9!res. 6!ch metho9s are time;cons!ming an9 care m!st be
ta@en to ens!re that am0licons acci9entally release9 into the laboratory
en>ironment 9o not contaminate the 1N8 0re0aration an9 clean rooms.
7eal;time PC7 machines greatly sim0li2y am0licon recognition by 0ro>i9ing the
means to monitor the acc!m!lation o2 s0eci2ic 0ro9!cts contin!o!sly 9!ring
cycling. 8ll c!rrent instr!ments 9esigne9 2or real;time PC7 meas!re the 0rogress
o2 am0li2ication by monitoring changes in 2l!orescence &ithin the PC7 t!be.
Changes in 2l!orescence can be lin@e9 to 0ro9!ct acc!m!lation by a >ariety o2
metho9s. 8 2!rther a9>antage o2 the real;time 2ormat is that the analysis can be
0er2orme9 &itho!t o0ening the t!be &hich can then be 9is0ose9 o2 &itho!t the
ris@ o2 9issemination o2 PC7 am0licons or other target molec!les into the
9
laboratory en>ironment. 8ltho!gh alternati>e metho9s 2or a>oi9ing PC7
contamination are a>ailable= containment &ithin the PC7 >essel is li@ely to be the
most e22icient an9 cost;e22ecti>e. 8 maIor 9ra&bac@ o2 stan9ar9 PC7 2ormats that
rely on en9;0oint analysis is that they are not B!antitati>e beca!se the 2inal yiel9
o2 0ro9!ct is not 0rimarily 9e0en9ent !0on the concentration o2 the target
seB!ence in the sam0le. 7eal;time PC7 o>ercomes this limitation.
2.4 Real-Time PCR Chemistries
:here are t&o general a00roaches !se9 to obtain a 2l!orescent signal 2rom the
synthesis o2 0ro9!ct in PC7. :he 2irst 9e0en9s !0on the 0ro0erty o2 2l!orescent
9yes s!ch as 6C?7 /reen I to bin9 to 9o!ble stran9e9 1N8 an9 !n9ergo a
con2ormational change that res!lt in an increase in their 2l!orescence. :he
secon9 a00roach is to !se 2l!orescent resonance energy trans2er ()7E:). :hese
metho9s !se a >ariety o2 means to alter the relati>e s0atial arrangement o2
0hoton 9onor an9 acce0tor molec!les. :hese molec!les are attache9 to 0robes=
0rimers or the PC7 0ro9!ct an9 are !s!ally selecte9 so that am0li2ication o2 a
s0eci2ic 1N8 seB!ence brings abo!t an increase in 2l!orescence at a 0artic!lar
&a>elength.
8 maIor a9>antage o2 the real;time PC7 instr!ments an9 signal trans9!ction
systems c!rrently a>ailable is that it is 0ossible to characteriAe the PC7 am0licon
in situ on the machine. :his is 9one by analysis o2 the melting tem0erat!re an93or
10
0robe hybri9isation characteristics o2 the am0licon &ithin the PC7 reaction
mi*t!re. In the intercalating 9ye system the melting tem0erat!re o2 the am0licon
can be estimate9 by meas!ring the le>el o2 2l!orescence emitte9 by the 9ye as
the tem0erat!re is increase9 2rom belo& to abo>e the e*0ecte9 melting
tem0erat!re. :he metho9s that rely !0on 0robe hybri9isation to 0ro9!ce a
2l!orescent signal are generally less liable to 0ro9!ce 2alse 0ositi>e res!lts than
alternati>e metho9s s!ch as the !se o2 intercalating 9yes to 9etect net synthesis
o2 9o!ble stran9e9 1N8 (9s1N8) 2ollo&e9 by melting analysis o2 the 0ro9!ct.
(ybri9isation= 7eson6ense an9 hy9rolysis 0robe systems gi>e 2l!orescent
signals that are only 0ro9!ce9 &hen the target seB!ence is am0li2ie9 an9 are
!nli@ely to gi>e 2alse 0ositi>e res!lts. 8n a99itional 2eat!re o2 the hybri9isation=
7eson6ense an9 relate9 metho9s is that it is also 0ossible to meas!re the
tem0erat!re at &hich the 0robes 9isassociate 2rom their com0lementary
seB!ences gi>ing 2!rther >eri2ication o2 the s0eci2icity o2 the am0li2ication
reaction. 8n im0ortant 2eat!re o2 many o2 the 0robe systems is that they are
com0atible &ith m!lti0le*ing 9!e to the a>ailability o2 2l!oro0hores &ith resol>able
emission s0ectra.
11
2.5 Quantification
"nli@e stan9ar9 PC7= real;time PC7 instr!ments meas!re the @inetics o2 0ro9!ct
acc!m!lation in each PC7 reaction t!be. /enerally= no 0ro9!ct is 9etecte9
9!ring the 2irst 2e& tem0erat!re cycles as the 2l!orescent signal is belo& the
9etection threshol9 o2 the instr!ment. (o&e>er= most combinations o2 machine
an9 2l!orescence re0orter are ca0able o2 9etecting the acc!m!lation o2
am0licons be2ore the en9 o2 the e*0onential am0li2ication 0hase. 1!ring this time
the e22iciency o2 PC7 is o2ten close to 100J gi>ing a 9o!bling o2 the B!antity o2
0ro9!ct at each cycle. 8s 0ro9!ct concentrations a00roach the nanogram 0er ml
le>el the e22iciency o2 am0li2ication 2alls 0rimarily beca!se the am0licons re;
associate 9!ring the annealing ste0. :his lea9s to a 0hase 9!ring &hich the
acc!m!lation o2 0ro9!ct is a00ro*imately linear &ith a constant le>el o2 net
synthesis at each cycle. )inally= a 0latea! is reache9 &hen net synthesis
a00ro*imates Aero. <!anti2ication in real;time PC7 is 9one by meas!ring the
n!mber o2 cycles reB!ire9 2or the 2l!orescent signal to reach a threshol9 le>el or
the secon9 9eri>ati>e ma*im!m o2 the 2l!orescence >ers!s cycle c!r>e. :his
cycle n!mber is 0ro0ortional to the n!mber o2 co0ies o2 tem0late in the sam0le.
12
2. Real-Time PCR Data Anal!sis
:he so2t&are 0ro>i9e9 &ith real;time PC7 instr!ments allo&s three 0rinci0le
ty0es o2 9ata analysis. 1) Meas!rement o2 the cycle n!mber at &hich any
increase in the 2l!orescence &ithin each reaction >essel reaches signi2icance. 2)
:he 9ata are !se9 in conI!nction &ith the res!lts 2rom e*ternal stan9ar9s to
estimate the original n!mber o2 tem0late co0ies. 4) Melting c!r>es are
trans2orme9 to 0ro>i9e 0lots o2 9)39: against : () K 2l!orescence an9
:Ktem0erat!re) in &hich a 0ea@ (melting 0ea@) occ!rs at the eB!ilibri!m
tem0erat!re 2or each 9!0le*. In general the 9i22erent so2t&are is easy to !se an9
allo&s ra0i9 an9 re0ro9!cible 9ata analysis.
13
2." Non-PCR A##lications
7eal;time PC7 machines are also ca0able o2 !se as real;time 2l!orimeters. )or
e*am0le= one sim0le a00lication is estimation o2 the melting tem0erat!re (:
m
) o2
an oligon!cleoti9e. :he oligon!cleoti9e is mi*e9 &ith its com0lementary
seB!ence in the 0resence o2 a 9ye s!ch as 6C?7 /reen I= the tem0erat!re is
increase9 an9 the le>el o2 2l!orescence is meas!re9 to gi>e a melting c!r>e 2rom
&hich the :
m
may be 9e9!ce9.
7eal;time PC7 0resents an alternati>e a00lication !sing a real;time PC7
instr!ment that relies on real;time 2l!orimetry. N86?8 is a metho9 2or the
isothermal am0li2ication o2 7N8 that 0ro9!ces B!antities o2 antisense 7N8
co0ies. Molec!lar beacons com0lementary to the 0ro9!ct are !se9 to gi>e a
2l!orescent signal.
14
3& E6E/'7ENTA8
3.1.A##aratus
1) 2 * 1;.mL 0i0ettes
2) 2 * 1;1000LL 0i0ettes=
4) 42 * 1.. ml >ials=
#) Centri2!gal machine=
.) Morte* machine=
$) 6tic@er labels=
+) Com0!teriAe9 7eal;7ime PC7 system.
3&)& Experimenta* proced#re
3.2.1. Dilution of each of the 4 sin$le stranded DNAs %3& 4& 5& '
1. :a@ing any ra& sam0le 1N8= e.g $4= ta@e o!t 1Ll o2 ra& (20ng3Ll) 1N8 $4 an9
% Ll o2 &ater res0ecti>ely= mi* them in a 1..ml >ial by centri2!ge an9 then Morte*.
Centri2!ging ens!res that the contents o2 the >ial 9o not cling to the &alls o2 the
>ial &hilst >orte* &ill ens!re &ell;mi*ing o2 the sam0le. :his ma@es a sam0le &ith
concentration 2.0ng3LL= &hich is a 10 times 9il!tion o2 the ra& 1N8 $4. Label
sam0le as $4;1.
2. :a@e 1Ll o2 sam0le $4;1 an9 %Ll o2 &ater res0ecti>ely= mi* them in a 1..ml >ial
by centri2!ge an9 then Morte*. :his gi>es a 0.2ng3LL 1N8 $4 sam0le= &hich is a
100 times 9il!tion o2 the ra& 1N8 $4. Label sam0le as $4;2.
15
4. 7e0eat ste0s 1 to 2 2or 1N8 sam0les $#= $. an9 $$. In total there &ill be 8
sam0les (# sam0les &ith concentration 2.0ng3LL= # sam0les &ith concentration o2
0.2ng3LL.)
3&)&) reparation of testin, samp*es in samp*e 5ia*s 9%&.m*:
3.2.2.1 (atched dou)le stranded DNA 3*4
1) Pre0are 4 i9entical sam0les &ith high concentration:
0..LL o2 $4;1 (2.0ng3Ll 1N8 $4) N 0..Ll o2 $#;1 (2.0ng3Ll 1N8 $#) N 11..Ll o2
(2F N 12..LL o2 6C?7. Mi* them by &ell in a 1..ml >ial by centri2!ge an9 then
Morte*. /ently trans2er (to a>oi9 b!bbles32oams) each sam0le (2.Ll) 2rom the
1..mL >ial into the 9e2ine9 &ell in the sam0le 0late (labele9 as 8124)
2) Pre0are 4 i9entical sam0les &ith lo& concentration:
0..LL o2 $4;2 (0.2ng3Ll 1N8 $4) N 0..Ll o2 $#;2 (0.2ng3Ll 1N8 $#) N 11..Ll o2
(2F N 12..LL o2 6C?7. /ently trans2er (to a>oi9 b!bbles32oams) each sam0le
(2.Ll) 2rom the 1..mL >ial into the 9e2ine9 &ell in the sam0le 0late (labele9 as
?124)
16
3.2.2.2 (ismatched dou)le stranded DNA 3*5
1) Pre0are 4 i9entical sam0les &ith high concentration:
0..LL o2 $4;1 (2.0ng3Ll 1N8 $4) N 0..Ll o2 $.;1 (2.0ng3Ll 1N8 $.) N 11..Ll o2
(2F N 12..LL o2 6C?7. Mi* them by &ell in a 1..ml >ial by centri2!ge an9 then
Morte*. /ently trans2er (to a>oi9 b!bbles32oams) each sam0le (2.Ll) 2rom the
1..mL >ial into the 9e2ine9 &ell in the sam0le 0late (labele9 as C124)
2) Pre0are 4 i9entical sam0les &ith lo& concentration:
0..LL o2 $4;2 (0.2ng3Ll 1N8 $4) N 0..Ll o2 $.;2 (0.2ng3Ll 1N8 $.) N 11..Ll o2
(2F N 12..LL o2 6C?7. /ently trans2er (to a>oi9 b!bbles32oams) each sam0le
(2.Ll) 2rom the 1..mL >ial into the 9e2ine9 &ell in the sam0le 0late (labele9 as
1124)
3.2.2.3 (ismatched dou)le stranded DNA 3*
1) Pre0are 4 i9entical sam0les &ith high concentration:
0..LL o2 $4;1 (2.0ng3Ll 1N8 $4) N 0..Ll o2 $$;1 (2.0ng3Ll 1N8 $$) N 11..Ll o2
(2F N 12..LL o2 6C?7. Mi* them by &ell in a 1..ml >ial by centri2!ge an9 then
Morte*. /ently trans2er (to a>oi9 b!bbles32oams) each sam0le (2.Ll) 2rom the
1..mL >ial into the 9e2ine9 &ell in the sam0le 0late (labele9 as E124)
17
2) Pre0are 4 i9entical sam0les &ith lo& concentration:
0..LL o2 $4;2 (0.2ng3Ll 1N8 $4) N 0..Ll o2 $$;2 (0.2ng3Ll 1N8 $$) N 11..Ll o2
(2F N 12..LL o2 6C?7. /ently trans2er (to a>oi9 b!bbles32oams) each sam0le
(2.Ll) 2rom the 1..mL >ial into the 9e2ine9 &ell in the sam0le 0late (labele9 as
)124)
3.2.2.4 Pre#are 3 identical +lan, -am#les
1) Each consisting o2 12..Ll o2 (2F N 12..LL o2 6C?7. /ently trans2er (to a>oi9
b!bbles32oams) each sam0le (2.Ll) 2rom the 1..mL >ial into the 9e2ine9 &ell in
the sam0le 0late (labele9 as blan@124)
3.2.2.5 Anal!.e sam#les usin$ Real-Time PCR s!stem
1) Place sam0le 0late in 7eal;:ime PC7 system an9 start 9iagnosis. Fbtaine9 all
reB!ire9 gra0hs.
18
(& /es#*ts
"sing the 7eal;:ime PC7= sam0les o2 1N8 $4= $#= $.= $$ an9 blan@s that &ere
0lace9 on a sam0le 0late &ere analyAe9. 8 gra0h o2 7)" (2l!orescent signal)
>ers!s tem0erat!re an9 the corres0on9ing -d(RFU)/dT >ers!s tem0erat!re gra0h
&ere obtaine9. In the a00en9i* at the en9 o2 the re0ort= tables 9etailing the
a00ro*imate melting 0oint tem0erat!res 2or the s0eci2ic hybri9s are sho&n. :he
-d(RFU)/dT >ers!s tem0erat!re gra0hs are !se9 to 9etermine the hybri9iAation
tem0erat!re 2or the 9i22erent sam0les. :hey are 0resente9 here as 2ollo&=
4.1 /ra#h for 0!)rid DNA 3 1ith 4 %sam#le A123& 0i$h Conc'
?l!e: 81= 7e9: 82= /reen: 84
)ig #.1 Melt C!r>e Pea@ Chart (;9)39t >s :) 2or sam0le 8124
Melting 0oint (a00ro*imately) K +8.. OC
19
4.2 /ra#h for 0!)rid DNA 3 1ith 4 %sam#le +123& 2o1 Conc'
?l!e: ?1= 7e9: ?2= /reen: ?4
)ig #.2 Melt C!r>e Pea@ Chart (;9)39t >s :) 2or sam0le ?124
Melting 0oint (a00ro*imately) K +8.. OC
20
4.3 /ra#h for 0!)rid DNA 3 1ith 5 %sam#le C123& 0i$h Conc'
?l!e: C1= 7e9: C2= /reen: C4
)ig #.4 Melt C!r>e Pea@ Chart (;9)39t >s :) 2or sam0le C124
Melting 0oint (a00ro*imately) K ++.. OC
21
4.4 /ra#h for 0!)rid DNA 3 1ith 5 %sam#le D123& 2o1 Conc'
?l!e: 11= 7e9: 12= /reen: 14
)ig #.# Melt C!r>e Pea@ Chart (;9)39t >s :) 2or sam0le 1124
Melting 0oint (a00ro*imately) K ++.. OC
22
4.5 /ra#h for 0!)rid DNA 3 1ith %sam#le 3123& 0i$h Conc'
?l!e: E1= 7e9: E2= /reen: E4
)ig #.. Melt C!r>e Pea@ Chart (;9)39t >s :) 2or sam0le E124
Melting 0oint (a00ro*imately) K +8.2 OC
23
4. /ra#h for 0!)rid DNA 3 1ith %sam#le 4123& 2o1 Conc'
?l!e: )1= 7e9: )2= /reen: )4
)ig #.$ Melt C!r>e Pea@ Chart (;9)39t >s :) 2or sam0le )124
Melting 0oint (a00ro*imately) K +8.2 OC
24
4." -am#le +lan,123
?l!e: ?lan@1= 7e9: ?lan@2= /reen: ?lan@4
)ig #.+ Melt C!r>e Pea@ Chart (;9)39t >s :) 2or sam0le ?lan@124
25
.& Disc#ssion
5.1 Comments on ho1 the DNA se5uence affects
h!)ridi.ation*deh!)ridisation
(ybri9isation is 9e2ine9 as the association o2 t&o com0lementary single;stran9e9
n!cleic aci9s by hy9rogen bon9s. :he hy9rogen bon9ing is base9 on s0eci2ic
base;0airing bet&een a 0!rine an9 a 0yrimi9ine so that the 1N8 molec!le can
maintain a 9o!ble heli* o2 2 nm 9iameter accor9ing to the -atson an9 Cric@
mo9el. It is interesting to note the g!anine an9 cytosine 2orms three hy9rogen
bon9s= &hereas a9enine an9 thymine 2orms t&o hy9rogen bon9s. )or the
re>erse case= 9ehybri9isation is the se0aration o2 the 0olyn!cleoti9e stran9s
!s!ally ca!se9 by the brea@ing o2 hy9rogen bon9s 9!e to heat or chemical
reagents.
In this e*0eriment= &e &ere 0ro>i9e9 &ith 2o!r single stran9e9 1N8 stran9s
namely $4= $#= $. an9 $$. 1N8 $4 an9 $# are com0letely com0lementary to
each other= hence are 0er2ectly matche9 accor9ing to Charga225s r!les. -here>er
one 1N8 stran9 has an 8= the 0artner stran9 has a :. 8n9 a / in one stran9 is
al&ays 0aire9 &ith a C in the com0lementary stran9.
Com0aring &ith 1N8 $#= 1N8 $. has a mismatch at the centre ca!se9 by a
single base s!bstit!tion o2 g!anine by cytosine. :he corres0on9ing n!cleoti9e on
1N8 $4 is cytosine. ?y Charga225s r!les= the com0lementary n!cleoti9e 2or
cytosine is g!anine.
26
6imilarly= 1N8 $$ has a mismatch at the en9 &here g!anine is re0lace9 be
cytosine. :h!s= 1N8 $4 an9 $$ cannot 2orm a com0lete 1N8 molec!le as the
cytosine at the 45 en9 o2 1N8 $$ cannot 2orm hy9rogen bon9s &ith the cytosine
at the .5 en9 o2 1N8 $4.
1!ring sam0le 0re0aration= 1N8 $4 &as mi*e9 &ith the other 4 com0lementary
single stran9s res0ecti>ely. (ybri9isation occ!rs as hy9rogen bon9s &ere 2orme9
bet&een the com0lementary bases 8;: an9 C;/= to 2orm 9o!ble stran9e9 1N8.
8ll 9o!ble;stran9e9 n!cleic aci9s ha>e a s0eci2ic melting tem0erat!re= &hich
9e0en9s mainly !0on their s0eci2ic g!anine;cytosine content. 6ince there are 4
hy9rogen bon9s bet&een g!anine an9 cytosine in base 0airing= a higher /;C
content &ill in9icate a higher melting tem0erat!re as there are more hy9rogen
bon9s to be bro@en. -hen mismatch occ!rs= 2e&er hy9rogen bon9s can be
2orme9 com0are9 to the 1N8 hybri9 &ith 0er2ectly com0lementary base;0airs.
:his res!lts in a 1N8 molec!le that is easier to 9enat!re an9 its melting 0oint
9ecreases.
In the sam0les o2 $43$. an9 $43$$= the re9!ction in the hy9rogen bon9s 9!e to
mismatch com0are9 to the com0letely matche9 $43$$ is eB!al. :here is a
re9!ction o2 4 hy9rogen bon9s as a g!anine is s!bstit!te9 by a cytosine. :he
signi2icant 9i22erence bet&een $43$. an9 $43$$ is in the location o2 the mismatchP
27
$43$. in the mi99le an9 $43$$ at the en9. :his 2actor &ill a22ect the melting 0oint
o2 the 1N8 hybri9.
:E6: 68MPLE :m at high
concentration (QC)
:m at lo&
concentration (QC)
1N8 $43$# +8.. +8..
1N8 $43$. ++.. ++..
1N8 $43$$ +8.2 +8.2
)rom the e*0erimental res!lts obtaine9= 1N8 $43$# has the highest melting 0oint
tem0erat!re b!t 1N8 $43$. has the lo&est melting 0oint tem0erat!re. In a99ition=
it is interesting to note that 1N8 $43$$ has a melting 0oint >ery close to the
0er2ectly matche9 1N8 hybri9 o2 $43$#.
-e can concl!9e that a mismatch at the en9 has the least e22ect on the melting
0oint tem0erat!reP hence the e22ect on hybri9iAation39ehybri9isation is minimal.
-hereas= a mismatch at the centre has a more 0rono!nce9 e22ect on the
hybri9iAation39ehybri9isation 0rocess as its melting 0oint 9e>iates the most 2rom
that o2 1N8 $43$#. :h!s= the 9i22erent location o2 a mismatch 9oes a22ect the
s!rro!n9ing base 0airs to a 9i22erent 9egree.
:he rea9ings recor9e9 2or the 1N8 hybri9s at lo& concentration 9o not ha>e a
9istinct 0ea@ 2or the melting 0oint tem0erat!re an9 ha>e n!mero!s small
28
2l!ct!ations o>er a &i9e tem0erat!re range. (ence= &e !se9 the >al!es obtaine9
2rom the high concentration sam0les.
I2 there are more mismatches in the 1N8 hybri9s= then the 9e>iation o2 the
melting 0oint tem0erat!re is e*0ecte9 to increase (:m 9ecreases by abo!t 1QC
2or e>ery 1J o2 mismatche9 base 0airs). :his is 9!e to the greater e22ect on
base;0airing 9!ring hybri9iAation. :here are t&o hy9rogen bon9s bet&een
a9enine an9 thymine &hich is lesser than that bet&een g!anine an9 cytosine.
:here2ore= a mismatch bet&een a9enine an9 thymine &ill a22ect hybri9iAation to a
smaller 9egree.
5.2 Comments on DNA len$th affectin$ the h!)ridi.ation*deh!)ridisation
#rocess
?esi9es the 9egree o2 seB!ence mismatch= the melting tem0erat!re is also
9e0en9ent !0on the length o2 the seB!ences to be hybri9iAe9P the shorter the
1N8 seB!ence= the lo&er the melting tem0erat!re. It there2ore ma@es sense to
ma*imise 1N8 length in or9er to minimise melting tem0erat!re re9!ction 9!e
both to length an9 9egree o2 seB!ence mismatch. -hen the 1N8 hybri9 is short=
its base com0osition &ill become the 0re9ominant 2actor.
In this e*0eriment= the 1N8 stran9s 0ro>i9e9 &ere 80 n!cleoti9es long. (o&e>er=
in the case o2 1N8 $. an9 $$= the mismatch 0ortions o2 the hybri9s 0ro>i9e the
a00ro*imation o2 the li@ely e22ect o2 ha>ing stran9s o2 shorter length. :his is
29
beca!se the n!mber o2 hy9rogen bon9s in a mismatche9 1N8 hybri9 is lesser
than that o2 a 0er2ect match.
5.3 The effect of NaCl concentration on h!)ridi.ation*deh!)ridisation
8 n!cleoti9e has a 0hos0hate gro!0 attache9 to the .5 carbon o2 9eo*yribose.
Notice also that the 0hos0hate gro!0 o2 one n!cleoti9e is attache9 to the 45
carbon o2 an a9Iacent carbon by a co>alent 0hos0ho9iester bon9. )!rther
con9ensation o2 n!cleoti9es &ill res!lt in a 1N8 stran9 &ith a s!gar;0hos0hate
bac@bone. 8s the 0hos0hate gro!0s are negati>ely charge9= the 1N8 stran9s
an9 their hybri9 helices &ill contain a large n!mber o2 negati>ely charge9
0hos0hate gro!0s. :hese gro!0s &ill res!lt in re0!lsion bet&een 1N8 single
stran9s an9 has to be o>ercome 9!ring hybri9iAation.
NaCl ioniAes in &ater to 2orm Na
N
ions that &ill ne!traliAe the negati>ely charge9
0hos0hate gro!0s at the s!gar;0hos0hate bac@bone o2 the 1N8 an9 re9!ce
re0!lsion bet&een the 1N8 stran9s 9!ring hybri9iAation. 8s a res!lt= the hybri9
1N8 is har9er to 9ehybri9ise an9 &ill ha>e a higher melting tem0erat!re.
:here2ore= NaCl concentration 9oes a22ect hybri9iAation39ehybri9isation. 8n
increase in NaCl concentration &ill also increase the melting tem0erat!re o2 the
1N8 hybri9 !ntil the sat!ration 0oint. :his >al!e o2 NaCl concentration gi>es the
ma*im!m melting tem0erat!re. 8ny 2!rther increase in NaCl concentration
beyon9 the sat!ration 0oint &ill not increase the melting tem0erat!re 2!rther.
30
-& Conc*#sion
In this e*0eriment= &e !se9 the real time B!anti2ication o2 the 2ormation o2 9o!ble
stran9e9 1N8 &hich is monitore9 at 9i22erent tem0erat!res. :his metho9 ha9
enable9 !s to !n9erstan9 &hen hybri9iAation39ehybri9isation occ!rs an9 ho&
seB!ence mismatch &o!l9 a22ect the same 0rocess.
?ase9 on the gra0hs in the res!lts section an9 the 1N8 seB!ences 2o!n9 in the
800en9i*= it can be seen that the 0resence o2 a mismatch in the 1N8 seB!ences
&ill a22ect the hybri9iAation39ehybri9isation 0rocess. )!rthermore= the melting
0oint o2 the hybri9 &ill 9ecrease i2 the 9egree o2 mismatch is greater. :he location
o2 the mismatch in the 1N8 hybri9 &ill also a22ect the
hybri9iAation39ehybri9isation 0rocess= &hereby a mismatch at the centre &ill
ha>e a more 0rono!nce9 e22ect than a mismatch at the en9s.
-e can concl!9e that a mismatch res!lts in 2e&er hy9rogen bon9s hence the
lo&er melting 0oint o2 the mismatche9 1N8 hybri9s.
-e also sa& the e22ect o2 1N8 length on hybri9iAation39ehybri9isation. :he
longer the length o2 1N8= the higher the melting 0oints o2 the hybri9s. (ence=
1N8 length an9 1N8 seB!encing are e*0ecte9 to ha>e a similar e22ect on the
hybri9iAation39ehybri9isation 0rocess.
31
In9ee9= NaCl 9oes in2l!ence the hybri9iAation39ehybri9isation 0rocess as Na
N
ions &ill ne!traliAe the negati>e charges o2 the s!gar;0hos0hate bac@bone o2 the
1N8 stran9s. :his &ill lo&er the re0!lsion bet&een stran9s 9!ring hybri9iAation
to 2orm a more stable hybri9 an9 a higher melting 0oint as &ell
:he obIecti>e o2 this e*0eriment &as to !n9erstan9 the im0ortance o2 seB!ence
com0lementary in 1N8 hybri9iAation. 8ltho!gh the mismatch bet&een g!anine
an9 cytosine &as analyAe9= &e co!l9 ha>e gras0e9 a 9ee0er analysis i2 the
mismatch bet&een a9enine an9 thymine &as also incl!9e9. :he mismatch
bet&een a9enine an9 thymine &o!l9 e*ert a less 0rono!nce e22ect on melting
0oint 9!e to the 2e&er hy9rogen bon9s in>ol>e9 in this base 0air.
1& /eferences
32
1) (ig!chi= 7.= )oc@ler= C.= 1ollinger= /.= an9 -atson= 7. ). 1%%4. Rinetic PC7:
7eal time monitoring o2 1N8 am0li2ication reactions. ?iotechnology 11:102$;
1040.
2) Nelson 1.L. an9 Co* M. M.= Lehninger Princi0les o2 ?iochemistry= #th E9.=
Cha0ter $= - ( )reeman S Co.= 200#.
4. Moet 1.= Moet '.= an9 Pratt C.= )!n9amentals o2 ?iochemisty= '.-iley S 6ons=
2001.
2& Notation
1N8 1eo*yribon!cleic 8ci9
PC7 Polymerase Chain 7eaction
<;PC7 <!antitati>e Polymerase Chain 7eaction
7)" 7elati>e )l!orescent "nit
33