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Department of Chemical & BiomolecularEngineering

THE NATIONALUNIVERIT! of

IN"A#ORE

Chemical Engineering Process Laboratory ICN2108

Experiment B3:

Thermodynamics and Kinetics of DNA Hybridization

Name (Matric No.): Chen !eling Cheryl ("0#$%#8r) Chie& 'ianao eli* ("0#$+#%h)

Ch!a ,he -ei ("0#$+8&)

/ro! : M2

ate o : $343200$E*eriment

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emonstrator5s :

6ignat!re /7E:

CONTENT A!E"#mmary$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$3

%& 'ntrod#ction$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$(

)& Theoretica* Bac+,ro#nd$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$-2.1 N2.2 n intro9!ction on the 7eal time PC72.4 he Nee9 or 7eal;ime PC72.# 7eal;ime PC7 Chemistries2.

2.$ 7eal;ime PC7 ata nalysis2.+ Non;PC7 lications

3&Experimenta*$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$%.4.1 arat!s4.2 E*erimental Proce9!re4.2.1. il!tion o each o the # single stran9e9 Ns ($4= $#= $= $$)4.2.2 Prearation o testing samles in samle >ials (1.ml)4.2.2.1 Matche9 9o!ble stran9e9 N $43$#4.2.2.2 Mismatche9 9o!ble stran9e9 N $43$4.2.2.4 Mismatche9 9o!ble stran9e9 N $43$$

4.2.2.# Preare 4 i9entical ?lan@ 6amles4.2.2. nalyAe samles !sing 7eal;ime PC7 system

(& /es#*ts$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$%0#.1 /rah or ybri9 N $4 &ith $# (samle 124= igh Conc)#.2 /rah or ybri9 N $4 &ith $# (samle ?124= Lo& Conc)#.4 /rah or ybri9 N $4 &ith $ (samle C124= igh Conc)#.# /rah or ybri9 N $4 &ith $ (samle 124= Lo& Conc)#. /rah or ybri9 N $4 &ith $$(samle E124= igh Conc)#.$ /rah or ybri9 N $4 &ith $$(samle 124= Lo& Conc)#.+ 6amle ?lan@124

.& Disc#ssion$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$)--& Conc*#sion$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$3%1& /eferences$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$332& Notation$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$33Appendix A /es#*ts tab*es

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"#mmary

-e are !sing the real time PC7 to in>estigate the eect o comlementary

seB!encing on N stability. his is 9one !sing N;$4= N;$#= N;$ an9

N;$$ at 9ierent concentrations !sing the henomena that 6?7 green

l!orescence 9ye intercalates &ith 9o!ble stran9e9 N. be seen that mismatch

o N seB!ences has an eect on the hybri9iAation39ehybri9iAation rocess=

&hereby the greater the 9egree o mismatch the lo&er the melting oint o the

hybri9. lso= mismatch at the centre o the hybri9 ten9s to ha>e a more

rono!nce9 eect on the rocess as comare9 to mismatch at the en9s.

ll these can be seen rom the grah o -d(RFU)/dT >ers!s temerat!re &hereby

mismatch at the center ten9 to ha>e the lo&est temerat!re.

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%&'ntrod#ction

-hen a 9o!ble stran9e9 N (or 9o!ble heli* N) is heate9 to 9enat!ration

temerat!re= the 9o!ble heli* &ill searate or Dmelt to orm t&o single stran9s.

he coole9 9o!ble helices &ill reorm (renat!ration) at regions o seB!ence

comlimentarity. his techniB!e is !se!l in 9etermining the seB!ence similarity in

Ns o 9ierent origin an9 the amo!nt o seB!ence reetition &ithin one stran9

o N.

ybri9iAation again 9een9s on the basic rincile o the n!mber o hy9rogen

bon9ing bet&een /C an9 bon9s in n!cleic aci9s. he rinciles in the

hybri9iAation stes are as ollo&:

1. N stran9s are searate9 by brea@ing the hy9rogen bon9s bet&een the

bases &ith heat to ro9!ce single stran9s.

2. 6tran9 searation is strictly 9een9ent !on t&o actors: he temerat!re

(amo!nt o heat energy) an9 the n!mber o hy9rogen bon9s (an incorrect

base airing 9oes not orm bon9s)

4. -hen the temerat!re 9ros= single stran9e9 N in the same sol!tion

that are comlementary sontaneo!sly come together by airing !

thro!gh their resecti>e an9 /C associations. he strength o their

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s!bseB!ent association 9een9s on the temerat!re an9 the total n!mber

o matching base airs.

Fne analytical techniB!e= the Polymerase Chain 7eaction (PC7)= has been

o!n9 to be e*tremely !se!l in 9etermining seB!ence similarity among Ns o

9ierent origins= as &ell as the amo!nt o seB!ence reetition &ithin one N.

he a9>ent o 7eal;ime PC7 has allo&e9 or more &i9esrea9 !se o PC7

assays or 9iagnostic !roses. In this e*eriment= 7eal;ime PC7 B!antiication

techniB!es &ill be emloye9 to monitor the ormation o 9o!ble;stran9e9 N at

9ierent temerat!res so as to !n9erstan9 &hen hybri9iAation an9

9ehybri9isation occ!rs an9 ho& they are aecte9 by a seB!ence mismatch.

PC7= ho&e>er= &ill not be carrie9 o!t.

Ob4ecti5e:

1. /ain an !n9erstan9ing o N hybri9iAation G the ormation o a 9o!ble

stran9e9 N oligon!cleoti9e molec!le rom t&o single stran9e9

molec!les.

2. "n9erstan9 the critical imortance o seB!ence comlementary in N

hybri9iAation.

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)& Theoretica* Bac+,ro#nd

2.1 DNA

eo*yribon!cleic aci9 (N) is the genetic material o all cells= carrying

inormation in the orm o a genetic co9e rom cell to cell an9 rom arent to

osring. gene is a segment o N that 9etermines the seB!ence o

nitrogeno!s bases in ribon!cleic aci9 (7N)= &hich in t!rn 9etermines the

seB!ence o amino aci9s in a seciic rotein. he characteristics o an organism

are 9etermine9 by the gene(s) an9 the res!lting rotein(s) that it is able to

synthesiAe. he roteins ro>i9e str!ct!re an9 catalyAe chemical reactions=

among other things. his constit!tes the central 9ogma o mo9ern biology= as

s!mmariAe9 belo&:

N N

Replication

N 7N P7FEIN

ranscrition ranslation

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o! can see that the lo& o genetic inormation rom N in>ol>es three main

stes:

(1) relication o the genetic inormation in N= &hich occ!rs in the synthesis

hase o interhase in the cell cycle= rior to cell 9i>ision=

(2) transcrition o N into 7Ns=

(4) translation o m7N into roteins.

N an9 7N are comose9 o chemical s!b!nits @no&n as n!cleoti9es. Each

n!cleoti9e is ma9e ! o a hoshate gro!= a entose s!gar= an9 one o the

o!r nitrogeno!s bases. N has o!r nitrogeno!s bases: a9enine= g!anine=

cytosine an9 thymine. 7N also is comose9 o o!r bases: a9enine= g!anine=

cytosine an9 !racil. he st!9ent sho!l9 remember that a9enine an9 g!anine are

H9o!ble ringH como!n9s categoriAe9 as !rines= &hereas cytosine= thymine an9

!racil are Hsingle ringH como!n9s categoriAe9 as yrimi9ines.

eo*yribon!cleoti9es contain the s!gar 9eo*yribose. 7ibon!cleoti9es= &hich are

the b!il9ing bloc@s or 7N synthesis= contain the s!gar ribose. he n!cleoti9es

are oine9 together by a single tye o hosho9iester lin@age to orm long chains

(stran9s) o N= &hich res!lts in the ormation o t&o stran9s arrange9 in a

9o!ble helical orm. In N= a9enine () is al&ays hy9rogen;bon9e9 to thymine

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() an9 g!anine (/) is al&ays hy9rogen;bon9e9 to cytosine (C). 6ince the t&o

stran9s o the N base;air &ith each other= they are sai9 to be comlementary

stran9s. his is @no&n as the r!le o base airing.

2.2 An introduction on the Real time PCR

he M' Mini gra9ient cycler oers:

thermal gra9ient eat!re that allo&s sim!ltaneo!s inc!bation at 8

9ierent temerat!res so yo! can otimiAe reactions or ma*im!m

eiciency an9 acc!rate B!antitation

7emar@ably rai9 arri>al at thermal !niormity= &hich ro9!ces the

recision nee9e9 or sensiti>e assays s!ch as B!antitati>e PC7

Peltier heat !m= b!ilt in;ho!se= that ro9!ces B!ic@ raming an9

acc!rate temerat!res to ens!re ast rero9!cible r!ns

#8;&ell samle bloc@ cae9 &ith an a9!stable heate9 li9 that

accommo9ates both !ll;height an9 lo&;roile t!bes an9 lates= so yo!

can r!n lo&;>ol!me reactions &ith minimal samle loss

e*t an9 grahical 9islay otions= &ith grahical e9iting or rai9ly

mo9iying rograms

Light &eight an9 comact siAe that allo& the instr!ment to it !st abo!t

any&here

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2.3 The Need for Real-Time PCR

M!ch o the technical eort in>ol>e9 in stan9ar9 PC7 is no& 9irecte9 to&ar9

ositi>e recognition o the amlicons. he imortant metho9s o ost;PC7

analysis rely on either the siAe or seB!ence o the amlicon. /el electrohoresis

is oten !se9 to meas!re the siAe o the amlicon an9 this is both ine*ensi>e

an9 simle to imlement. "nort!nately= siAe analysis has limite9 seciicity since

9ierent molec!les o aro*imately the same molec!lar &eight cannot be

9isting!ishe9. ConseB!ently= gel electrohoresis alone is not a s!icient PC7

en9;oint in many instances= incl!9ing most clinical alications.

Characterisation o the ro9!ct by its seB!ence is ar more reliable an9

inormati>e. Probe hybri9isation assays or this !rose are a>ailable b!t many

are m!lti;ste roce9!res. 6!ch metho9s are time;cons!ming an9 care m!st be

ta@en to ens!re that amlicons acci9entally release9 into the laboratory

en>ironment 9o not contaminate the N rearation an9 clean rooms.

7eal;time PC7 machines greatly simliy amlicon recognition by ro>i9ing the

means to monitor the acc!m!lation o seciic ro9!cts contin!o!sly 9!ring

cycling. ll c!rrent instr!ments 9esigne9 or real;time PC7 meas!re the rogress

o amliication by monitoring changes in l!orescence &ithin the PC7 t!be.

Changes in l!orescence can be lin@e9 to ro9!ct acc!m!lation by a >ariety o

metho9s. !rther a9>antage o the real;time ormat is that the analysis can be

erorme9 &itho!t oening the t!be &hich can then be 9isose9 o &itho!t the

ris@ o 9issemination o PC7 amlicons or other target molec!les into the

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laboratory en>ironment. ltho!gh alternati>e metho9s or a>oi9ing PC7

contamination are a>ailable= containment &ithin the PC7 >essel is li@ely to be the

most eicient an9 cost;eecti>e. maor 9ra&bac@ o stan9ar9 PC7 ormats that

rely on en9;oint analysis is that they are not B!antitati>e beca!se the inal yiel9

o ro9!ct is not rimarily 9een9ent !on the concentration o the target

seB!ence in the samle. 7eal;time PC7 o>ercomes this limitation.

2.4 Real-Time PCR Chemistries

here are t&o general aroaches !se9 to obtain a l!orescent signal rom the

synthesis o ro9!ct in PC7. he irst 9een9s !on the roerty o l!orescent

9yes s!ch as 6?7 /reen I to bin9 to 9o!ble stran9e9 N an9 !n9ergo a

conormational change that res!lt in an increase in their l!orescence. he

secon9 aroach is to !se l!orescent resonance energy transer (7E). hese

metho9s !se a >ariety o means to alter the relati>e satial arrangement o

hoton 9onor an9 accetor molec!les. hese molec!les are attache9 to robes=

rimers or the PC7 ro9!ct an9 are !s!ally selecte9 so that amliication o a

seciic N seB!ence brings abo!t an increase in l!orescence at a artic!lar

&a>elength.

maor a9>antage o the real;time PC7 instr!ments an9 signal trans9!ction

systems c!rrently a>ailable is that it is ossible to characteriAe the PC7 amlicon

in situon the machine. his is 9one by analysis o the melting temerat!re an93or

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robe hybri9isation characteristics o the amlicon &ithin the PC7 reaction

mi*t!re. In the intercalating 9ye system the melting temerat!re o the amlicon

can be estimate9 by meas!ring the le>el o l!orescence emitte9 by the 9ye as

the temerat!re is increase9 rom belo& to abo>e the e*ecte9 melting

temerat!re. he metho9s that rely !on robe hybri9isation to ro9!ce a

l!orescent signal are generally less liable to ro9!ce alse ositi>e res!lts than

alternati>e metho9s s!ch as the !se o intercalating 9yes to 9etect net synthesis

o 9o!ble stran9e9 N (9sN) ollo&e9 by melting analysis o the ro9!ct.

ybri9isation= 7eson6ense an9 hy9rolysis robe systems gi>e l!orescent

signals that are only ro9!ce9 &hen the target seB!ence is amliie9 an9 are

!nli@ely to gi>e alse ositi>e res!lts. n a99itional eat!re o the hybri9isation=

7eson6ense an9 relate9 metho9s is that it is also ossible to meas!re the

temerat!re at &hich the robes 9isassociate rom their comlementary

seB!ences gi>ing !rther >eriication o the seciicity o the amliication

reaction. n imortant eat!re o many o the robe systems is that they are

comatible &ith m!ltile*ing 9!e to the a>ailability o l!orohores &ith resol>able

emission sectra.

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2.5 Quantification

"nli@e stan9ar9 PC7= real;time PC7 instr!ments meas!re the @inetics o ro9!ct

acc!m!lation in each PC7 reaction t!be. /enerally= no ro9!ct is 9etecte9

9!ring the irst e& temerat!re cycles as the l!orescent signal is belo& the

9etection threshol9 o the instr!ment. o&e>er= most combinations o machine

an9 l!orescence reorter are caable o 9etecting the acc!m!lation o

amlicons beore the en9 o the e*onential amliication hase. !ring this time

the eiciency o PC7 is oten close to 100J gi>ing a 9o!bling o the B!antity o

ro9!ct at each cycle. s ro9!ct concentrations aroach the nanogram er ml

le>el the eiciency o amliication alls rimarily beca!se the amlicons re;

associate 9!ring the annealing ste. his lea9s to a hase 9!ring &hich the

acc!m!lation o ro9!ct is aro*imately linear &ith a constant le>el o net

synthesis at each cycle. inally= a latea! is reache9 &hen net synthesis

aro*imates Aero.

n!mber o cycles reB!ire9 or the l!orescent signal to reach a threshol9 le>el or

the secon9 9eri>ati>e ma*im!m o the l!orescence >ers!s cycle c!r>e. his

cycle n!mber is roortional to the n!mber o coies o temlate in the samle.

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2. Real-Time PCR Data Anal!sis

he sot&are ro>i9e9 &ith real;time PC7 instr!ments allo&s three rincile

tyes o 9ata analysis. 1) Meas!rement o the cycle n!mber at &hich any

increase in the l!orescence &ithin each reaction >essel reaches signiicance. 2)

he 9ata are !se9 in con!nction &ith the res!lts rom e*ternal stan9ar9s to

estimate the original n!mber o temlate coies. 4) Melting c!r>es are

transorme9 to ro>i9e lots o 939 against ( K l!orescence an9

Ktemerat!re) in &hich a ea@ (melting ea@) occ!rs at the eB!ilibri!m

temerat!re or each 9!le*. In general the 9ierent sot&are is easy to !se an9

allo&s rai9 an9 rero9!cible 9ata analysis.

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2." Non-PCR A##lications

7eal;time PC7 machines are also caable o !se as real;time l!orimeters. or

e*amle= one simle alication is estimation o the melting temerat!re ( m) o

an oligon!cleoti9e. he oligon!cleoti9e is mi*e9 &ith its comlementary

seB!ence in the resence o a 9ye s!ch as 6?7 /reen I= the temerat!re is

increase9 an9 the le>el o l!orescence is meas!re9 to gi>e a melting c!r>e rom

&hich the mmay be 9e9!ce9.

7eal;time PC7 resents an alternati>e alication !sing a real;time PC7

instr!ment that relies on real;time l!orimetry. N6? is a metho9 or the

isothermal amliication o 7N that ro9!ces B!antities o antisense 7N

coies. Molec!lar beacons comlementary to the ro9!ct are !se9 to gi>e a

l!orescent signal.

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4. 7eeat stes 1 to 2 or N samles $#= $ an9 $$. In total there &ill be 8

samles (# samles &ith concentration 2.0ng3L= # samles &ith concentration o

0.2ng3L.)

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) Preare 4 i9entical samles &ith high concentration:

0.L o $4;1 (2.0ng3l N $4) 0.l o $#;1 (2.0ng3l N $#) 11.l o

2F 12.L o 6?7. Mi* them by &ell in a 1.ml >ial by centri!ge an9 then

orte*. /ently transer (to a>oi9 b!bbles3oams) each samle (2l) rom the

1.mL >ial into the 9eine9 &ell in the samle late (labele9 as 124)

2) Preare 4 i9entical samles &ith lo& concentration:

0.L o $4;2 (0.2ng3l N $4) 0.l o $#;2 (0.2ng3l N $#) 11.l o

2F 12.L o 6?7. /ently transer (to a>oi9 b!bbles3oams) each samle

(2l) rom the 1.mL >ial into the 9eine9 &ell in the samle late (labele9 as

?124)

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3.2.2.2 (ismatched dou)le stranded DNA 3*5

1) Preare 4 i9entical samles &ith high concentration:

0.L o $4;1 (2.0ng3l N $4) 0.l o $;1 (2.0ng3l N $) 11.l o

2F 12.L o 6?7. Mi* them by &ell in a 1.ml >ial by centri!ge an9 then

orte*. /ently transer (to a>oi9 b!bbles3oams) each samle (2l) rom the

1.mL >ial into the 9eine9 &ell in the samle late (labele9 as C124)

2) Preare 4 i9entical samles &ith lo& concentration:

0.L o $4;2 (0.2ng3l N $4) 0.l o $;2 (0.2ng3l N $) 11.l o

2F 12.L o 6?7. /ently transer (to a>oi9 b!bbles3oams) each samle

(2l) rom the 1.mL >ial into the 9eine9 &ell in the samle late (labele9 as

124)

3.2.2.3 (ismatched dou)le stranded DNA 3*

1) Preare 4 i9entical samles &ith high concentration:

0.L o $4;1 (2.0ng3l N $4) 0.l o $$;1 (2.0ng3l N $$) 11.l o

2F 12.L o 6?7. Mi* them by &ell in a 1.ml >ial by centri!ge an9 then

orte*. /ently transer (to a>oi9 b!bbles3oams) each samle (2l) rom the

1.mL >ial into the 9eine9 &ell in the samle late (labele9 as E124)

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(& /es#*ts

"sing the 7eal;ime PC7= samles o N $4= $#= $= $$ an9 blan@s that &ere

lace9 on a samle late &ere analyAe9. grah o 7" (l!orescent signal)

>ers!s temerat!re an9 the correson9ing -d(RFU)/dT >ers!s temerat!re grah

&ere obtaine9. In the aen9i* at the en9 o the reort= tables 9etailing the

aro*imate melting oint temerat!res or the seciic hybri9s are sho&n. he

-d(RFU)/dT >ers!s temerat!re grahs are !se9 to 9etermine the hybri9iAation

temerat!re or the 9ierent samles. hey are resente9 here as ollo&=

4.1 /ra#h for 0!)rid DNA 3 ith 4 %sam#le A123& 0i$h Conc'

?l!e: 1= 7e9: 2= /reen: 4

ig #.1 Melt C!r>e Pea@ Chart (;939t >s ) or samle 124

Melting oint (aro*imately) K +8. OC

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4.2 /ra#h for 0!)rid DNA 3 ith 4 %sam#le +123& o Conc'

?l!e: ?1= 7e9: ?2= /reen: ?4

ig #.2 Melt C!r>e Pea@ Chart (;939t >s ) or samle ?124

Melting oint (aro*imately) K +8. OC

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4.3 /ra#h for 0!)rid DNA 3 ith 5 %sam#le C123& 0i$h Conc'

?l!e: C1= 7e9: C2= /reen: C4

ig #.4 Melt C!r>e Pea@ Chart (;939t >s ) or samle C124

Melting oint (aro*imately) K ++. OC

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4.4 /ra#h for 0!)rid DNA 3 ith 5 %sam#le D123& o Conc'

?l!e: 1= 7e9: 2= /reen: 4

ig #.# Melt C!r>e Pea@ Chart (;939t >s ) or samle 124

Melting oint (aro*imately) K ++. OC

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4.5 /ra#h for 0!)rid DNA 3 ith %sam#le 123& 0i$h Conc'

?l!e: E1= 7e9: E2= /reen: E4

ig #. Melt C!r>e Pea@ Chart (;939t >s ) or samle E124

Melting oint (aro*imately) K +8.2 OC

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4. /ra#h for 0!)rid DNA 3 ith %sam#le 123& o Conc'

?l!e: 1= 7e9: 2= /reen: 4

ig #.$ Melt C!r>e Pea@ Chart (;939t >s ) or samle 124

Melting oint (aro*imately) K +8.2 OC

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4." am#le +lan,123

?l!e: ?lan@1= 7e9: ?lan@2= /reen: ?lan@4

ig #.+ Melt C!r>e Pea@ Chart (;939t >s ) or samle ?lan@124

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6imilarly= N $$ has a mismatch at the en9 &here g!anine is relace9 be

cytosine. h!s= N $4 an9 $$ cannot orm a comlete N molec!le as the

cytosine at the 45 en9 o N $$ cannot orm hy9rogen bon9s &ith the cytosine

at the 5 en9 o N $4.

!ring samle rearation= N $4 &as mi*e9 &ith the other 4 comlementary

single stran9s resecti>ely. ybri9isation occ!rs as hy9rogen bon9s &ere orme9

bet&een the comlementary bases ; an9 C;/= to orm 9o!ble stran9e9 N.

ll 9o!ble;stran9e9 n!cleic aci9s ha>e a seciic melting temerat!re= &hich

9een9s mainly !on their seciic g!anine;cytosine content. 6ince there are 4

hy9rogen bon9s bet&een g!anine an9 cytosine in base airing= a higher /;C

content &ill in9icate a higher melting temerat!re as there are more hy9rogen

bon9s to be bro@en. -hen mismatch occ!rs= e&er hy9rogen bon9s can be

orme9 comare9 to the N hybri9 &ith erectly comlementary base;airs.

his res!lts in a N molec!le that is easier to 9enat!re an9 its melting oint

9ecreases.

In the samles o $43$ an9 $43$$= the re9!ction in the hy9rogen bon9s 9!e to

mismatch comare9 to the comletely matche9 $43$$ is eB!al. here is a

re9!ction o 4 hy9rogen bon9s as a g!anine is s!bstit!te9 by a cytosine. he

signiicant 9ierence bet&een $43$ an9 $43$$ is in the location o the mismatch

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$43$ in the mi99le an9 $43$$ at the en9. his actor &ill aect the melting oint

o the N hybri9.

E6 6MPLE m at high

concentration (QC)

m at lo&

concentration (QC)

N $43$# +8. +8.

N $43$ ++. ++.

N $43$$ +8.2 +8.2

rom the e*erimental res!lts obtaine9= N $43$# has the highest melting oint

temerat!re b!t N $43$ has the lo&est melting oint temerat!re. In a99ition=

it is interesting to note that N $43$$ has a melting oint >ery close to the

erectly matche9 N hybri9 o $43$#.

-e can concl!9e that a mismatch at the en9 has the least eect on the melting

oint temerat!re hence the eect on hybri9iAation39ehybri9isation is minimal.

-hereas= a mismatch at the centre has a more rono!nce9 eect on the

hybri9iAation39ehybri9isation rocess as its melting oint 9e>iates the most rom

that o N $43$#. h!s= the 9ierent location o a mismatch 9oes aect the

s!rro!n9ing base airs to a 9ierent 9egree.

he rea9ings recor9e9 or the N hybri9s at lo& concentration 9o not ha>e a

9istinct ea@ or the melting oint temerat!re an9 ha>e n!mero!s small

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l!ct!ations o>er a &i9e temerat!re range. ence= &e !se9 the >al!es obtaine9

rom the high concentration samles.

I there are more mismatches in the N hybri9s= then the 9e>iation o the

melting oint temerat!re is e*ecte9 to increase (m 9ecreases by abo!t 1QC

or e>ery 1J o mismatche9 base airs). his is 9!e to the greater eect on

base;airing 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.

hereore= a mismatch bet&een a9enine an9 thymine &ill aect hybri9iAation to a

smaller 9egree.

5.2 Comments on DNA len$th affectin$ the h!)ridiation*deh!)ridisation

#rocess

?esi9es the 9egree o seB!ence mismatch= the melting temerat!re is also

9een9ent !on the length o the seB!ences to be hybri9iAe9 the shorter the

N seB!ence= the lo&er the melting temerat!re. It thereore ma@es sense to

ma*imise N length in or9er to minimise melting temerat!re re9!ction 9!e

both to length an9 9egree o seB!ence mismatch. -hen the N hybri9 is short=

its base comosition &ill become the re9ominant actor.

In this e*eriment= the N stran9s ro>i9e9 &ere 80 n!cleoti9es long. o&e>er=

in the case o N $ an9 $$= the mismatch ortions o the hybri9s ro>i9e the

aro*imation o the li@ely eect o ha>ing stran9s o shorter length. his is

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beca!se the n!mber o hy9rogen bon9s in a mismatche9 N hybri9 is lesser

than that o a erect match.

5.3 The effect of NaCl concentration on h!)ridiation*deh!)ridisation

n!cleoti9e has a hoshate gro! attache9 to the 5 carbon o 9eo*yribose.

Notice also that the hoshate gro! o one n!cleoti9e is attache9 to the 45

carbon o an a9acent carbon by a co>alent hosho9iester bon9. !rther

con9ensation o n!cleoti9es &ill res!lt in a N stran9 &ith a s!gar;hoshate

bac@bone. s the hoshate gro!s are negati>ely charge9= the N stran9s

an9 their hybri9 helices &ill contain a large n!mber o negati>ely charge9

hoshate gro!s. hese gro!s &ill res!lt in re!lsion bet&een N single

stran9s an9 has to be o>ercome 9!ring hybri9iAation.

NaCl ioniAes in &ater to orm Naions that &ill ne!traliAe the negati>ely charge9

hoshate gro!s at the s!gar;hoshate bac@bone o the N an9 re9!ce

re!lsion bet&een the N stran9s 9!ring hybri9iAation. s a res!lt= the hybri9

N is har9er to 9ehybri9ise an9 &ill ha>e a higher melting temerat!re.

hereore= NaCl concentration 9oes aect hybri9iAation39ehybri9isation. n

increase in NaCl concentration &ill also increase the melting temerat!re o the

N hybri9 !ntil the sat!ration oint. his >al!e o NaCl concentration gi>es the

ma*im!m melting temerat!re. ny !rther increase in NaCl concentration

beyon9 the sat!ration oint &ill not increase the melting temerat!re !rther.

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-& Conc*#sion

In this e*eriment= &e !se9 the real time B!antiication o the ormation o 9o!ble

stran9e9 N &hich is monitore9 at 9ierent temerat!res. his metho9 ha9

enable9 !s to !n9erstan9 &hen hybri9iAation39ehybri9isation occ!rs an9 ho&

seB!ence mismatch &o!l9 aect the same rocess.

?ase9 on the grahs in the res!lts section an9 the N seB!ences o!n9 in the

en9i*= it can be seen that the resence o a mismatch in the N seB!ences

&ill aect the hybri9iAation39ehybri9isation rocess. !rthermore= the melting

oint o the hybri9 &ill 9ecrease i the 9egree o mismatch is greater. he location

o the mismatch in the N hybri9 &ill also aect the

hybri9iAation39ehybri9isation rocess= &hereby a mismatch at the centre &ill

ha>e a more rono!nce9 eect than a mismatch at the en9s.

-e can concl!9e that a mismatch res!lts in e&er hy9rogen bon9s hence the

lo&er melting oint o the mismatche9 N hybri9s.

-e also sa& the eect o N length on hybri9iAation39ehybri9isation. he

longer the length o N= the higher the melting oints o the hybri9s. ence=

N length an9 N seB!encing are e*ecte9 to ha>e a similar eect on the

hybri9iAation39ehybri9isation rocess.

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1) ig!chi= 7.= oc@ler= C.= ollinger= /.= an9 -atson= 7. . 1%%4. Rinetic PC7:

7eal time monitoring o N amliication reactions. ?iotechnology 11:102$;

1040.

2) Nelson .L. an9 Co* M. M.= Lehninger Princiles o ?iochemistry= #th E9.=

Chater $= - reeman S Co.= 200#.

4. oet .= oet '.= an9 Pratt C.= !n9amentals o ?iochemisty= '.-iley S 6ons=

2001.

2& Notation

N eo*yribon!cleic ci9

PC7 Polymerase Chain 7eaction

7" 7elati>e l!orescent "nit