Anil Nucleic Acids

8/12/2019 Anil Nucleic Acids

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Nucleic Acids

MCD Year 1

Anil Chopra

Contents

Contents................................................................................................................................. 1

Nucleic Acids 1 Nucleic Acids and Chromosomes...............................................................2

Nucleic acids.......................................................................................................................2

............................................................................................................................................... 4

Melting and re-annealing........................................................................................................ 4

Packaging of DNA into a chromosome...................................................................................5

Nucleic Acids 2 DNA e!lication" the Cell C#cle and Mitosis..............................................$

Nucleic Acids % - &ene 'rganisation and (ranscri!tion 1.....................................................1%

Nucleic Acids 4 &ene 'rganisation and (ranscri!tion ))....................................................1$

Nucleic Acids 5 - Protein translation and translational modification......................................1*

Nucleic Acids + Anal#sis of DNA......................................................................................2%


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Nucleic Acids 1 Nucleic Acids and ChromosomesAlex Perkins

1. Draw the structure of a nucleotide labelling the sugar, base and phosphates andexplain the difference between a nucleotide and a nucleoside.

Nucleic acids

DNA and RNA are nucleic acids molecules of heredit.

!he are macromolecules made up of a large number of nucleotides.

A nucleotide is composed of a base, a sugar, and a phosphate group.

A nucleoside is composed of a base and a sugar "no phosphate#.

!he sugar in DNA is deoxribose, the sugar in RNA is ribose.

Nucleotide$ deoxadenosine %&'triphosphate "dA!P#

A nucleoside is a nucleotide without the phosphate

(. )ist the bases found in DNA and RNA and indicate which ones are purines and which

ones are primidines.

DNA and RNA bases:

Primidines$

__

H

O

H

HO

H

C H

H

O1

23

4

5

2

N

N N

N

NH

H

H

PO

O

O

PO

O

O

PO

O

O

_

_

sugar deo,#riose in DNA

- riose in NA

ase!hos!hate

Cytosine

(C)

Uracil

(U)RNA only

Thyine

(T)!NA only

N

N

O

O

H

H

HN

N

O

O

CH3

H

HN

N

NH2

O H

H


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Purines$

The nucleosides:"deox#ctidine, "deox#thmidine, "deox#uridine, "deox#adenosine,

"deox#guanosine

The nucleotides:deoxadenosine %&'triphosphate "dA!P#* adenosine monophosphate"A+P# etc.

.

. Describe a single DNA chain and explain the difference between the %& and & ends.

Draw the structure of the double'stranded helix of DNA "not atomic structure#

showing base'pairing, the ma-or and minor grooes, and the directionalit of the

chains.

DNAis a long chain of deoxribose units linked b phosphodiester links.

!he phosphate on the %& carbon is linked to the /0 on the & carbon along the chain.

/n each deoxribose there is a base. !he chain has two ends. !he %& end and the & end. t is not smmetrical.

Adenine

A/

&uanine

&/

N

N N

N

N 0 2

0

0

N

N N

'

02

N

0

minor groove

major groove

The DNA double helix

"

O

O

H

H

H

N

H

N

N

N

O

N 2N

O

O

O

PO O

PO O

O

PO O

O

N

NN

N

NH2

O

H

O

N

NH2

O

5#

3#

3#

3#

5#

5#

OH

"

"

5' end

3' end

A single DNA chain


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!he primar se2uence is the linear se2uence of the bases. 3 conention, the nucleotide

se2uence is specified in the %& to & direction.

!he secondar structureof DNA is a right'handed double helix. !he two chains in the

helix run in opposite directions.

!he deoxribose and phosphate groups run along the outside of the helix, with the

negatie charges outside.

!he bases point inwards and the flat planes are perpendicular to the helix.

!he two chains are held together b hdrogen bonds between the bases.

!he two strands are complementar in their se2uence due to the specificit of base'

pairing. Adenine alwas pairs with !hmine* 4uanine alwas pairs with 5tosine.

6. Describe melting and re'annealing of complementar strands and what is meant b

7atson'5rick base'pairing.

A$enine

(A)

Thyine

(T)

%&anine

(%)

Cytosine

(C)

3 hy$ro'en on$sore stale

2 hy$ro'en on$sless stale

WatsonCric! base "airs

N

N

N

O

H

H

N

N

N

N O

N

H

H

H

N

N

N

N N

N

N

O

O

CH3

H

H

H

Melting and re-annealing

0igh temperature and8or low salt concentration causes the two strands to melt or

disassociate.

f ou then lower the temperature or increase the salt concentration, the two melted

strands will re'anneal into a double helix.

0bridisation$ in a mixture of DNA with different se2uences, the complementar strands

will find each other in the mixture.

%. 5ompare the genomes ofE.coliandHomo Sapiens.The E.coli genome

E.coli has 6.9 x 1:;base pairs in a single circular double'stranded molecule.

!he length of the


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A diploid cell has 6; chromosomes* (( pairs of >normal& chromosomes and ( sex

chromosomes.

;. Draw a diagram illustrating the packaging of DNA into nucleosomes and relate this to

chromosome structure.

Packaging of eukaryotic DNA

!he DNA in a diploid human cell is nearl ( m long. !o fit into cells, the DNA is tightl

packaged into chromatin.

5hromatin consists of DNA and proteins.

!he lowest leel of packaging is the nucleosome, which consists of DNA wrapped around

histone proteins.

!he nucleosomes form a chain, which pack into a helical arra.

Packaging of DNA into a chromosome

As the DNA has alread replicated, there are two identical copies and two identical

chromatids "sister chromatids# for each chromosome at metaphase.

in*er !NA

Ao&t 2++ ase ,airs o- !NA .ra,,e$ aro&n$the histones (core !NA)

Histone 1 et.een then&cleosoes

/ histones0 2 each o- 2A 2 3 an$ 4

A nucleosome

hort re'ion o- !NA $o&le heli4

ea$s on a strin' -or o- chroatin

3+ n chroatin -ier o- ,ac*e$

n&cleosoes

ection o- chroosoe in e4ten$e$

-or

Con$ense$ section o- eta,hase

chroosoe

5ntire eta,hase chroosoe

consistin' o- t.o sister chroati$s

Net res&lt0 each !NA olec&le

has een ,ac*a'e$ into a itotic

chroosoe that is 1+ +++ -ol$

shorter than its e4ten$e$ len'th


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9. Describe the human karotpe.

A karotpe is an organised profile of someone&s chromosomes.

A diploid human cell has 6; chromosomes

' (( pairs of >normal& chromosomes "autosomes#' ( sex chromosomes "? and @#

' ex chromosomes$ ?? for female* ?@ for male

G-banded karyotype of a Normal Male ell

!wo homologues of each chromosome


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Nucleic Acids # DNA $e"lication% the Cell C&cle and'itosis

Anil Cho!ra

1. ,!lain semi-conseratie re!lication.

Ne3 co!ies of DNA made should e identical.

ach daughter cell inherits one older strand and one ne3 strand.

ach strand seres as a tem!late.

2. Descrie the reaction catal#sed # DNA !ol#merases.DNA Helicase uses A(P to reak the h#drogen onds et3een the ase!airs to un6i!7 the DNA.

DNA Polymerase s#nthesises ne3 DNA # adding nucleotides to the%8 !rime/ end of the gro3ing chain.

%. Descrie ho3 nucleoside analogs can e used as drugs.9ome nucleoside analogs can e used as drugs ecause the#terminate chains. .g.

Ac#cloirused in treatment for 0er!es

C#tosine arainoseused in chemothera!#

Dideo,#c#tosine ddC/0): drug

A6idoth#midine A;(/0): drug.

4. Descrie the functions of the com!onents of the re!lication com!le, in !rokar#otesincluding the terms tem!late" !rimer" leading strand" lagging strand" 'kasaki fragmentand re!lication fork.

DNA !ol#merases need a templateand a primer.

(he re!lication !rocess egins at discrete !oints and occurs i-directionall#. ITALWAYS OCCURS FRO !" TO #"$

'n the lea%in& stran%continuous s#nthesis

'n the la&&in& stran%discontinuous s#nthesis

1. DNA helicase un3inds heli,.2.


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%. DNA !ol#merase ))) continuall# adds deo,#nucleotide!hos!hates onto the end of the !rimer to form the chaine,tension. (his re>uires A(P.

4. 'n the lagging strand there are man# NA !rimers thatare added to arious !oints on the tem!late strand.(heses are acted on # DNA !ol#merase ))) to !roduceO'a(a'i )ra&ments. (his also re>uires A(P. (helagging strand loo!s around so that the DNA!ol#merase ))) can 3ork on oth strands at thesame time.

5. uires a !roof-reading

mechanism to ensure no mistakes are made.


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Mutations changes in DNA se>uence/ are er# dangerous to the organism. An# errors in

re!lication cannot e re!aired.

DNA re!lication has an error fre>uenc# of aout 1 change !er 1= * ase !airs.

?efore a ne3 nucleotide is added" the !reious nucleotide is checked for

correct ase-!airing. )n errors 3ith DNA s#nthesis" the nucleotide 3ith the

incorrect ase 3ill not !ro!erl# fit and ond 3ith its corres!onding ase!air. (he !hos!hodiester ond et3een then nucleotides/ is thenh#drol#sed # the e*on+clease acti,ityof DNA!ol#merase ))) and the correct ase is added.

)n errors 3ith NA !rimers" the NA !rimer is

sim!l# remoed and re!laced 3ith accurate DNAs#nthesis.

+. Dra3 a diagram sho3ing re!lication of the .coli

chromosome.(he .coli chromosome is circular. (here is a single uni>ue

!oint of origin 'ri.C/ from 3here the t3o re!lication forks occur simultaneousl# in o!!osite

directions. (he t3o forks meet at the other side of the chromosome. e!lication of the .coli

chromosome uses different en6#mes.

$. Descrie the re!lication of mammalianchromosomes.

Mammalian chromosomes are long and linear.

e!lication originates at arious different !oints along


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the DNA at interals of around 1==kiloase !airs and !roceeds i-directionall#. )t finishes 3hen all the

forks hae met.

(here are at least 5 different DNA !ol#merases

Pol as#nthesises the !rimers for oth leading and lagging strands

Pol -is e>uialent to Pol ) in .coli

Pol %s#nthesises the leading strand

Pol es#nthesises the lagging strand

Pol &re!licates mitochondrial DNA

. Descrie the different !hases of the cellc#cle.

phase.itosisB cell diisionB 1hr. (his is 3here the 2

chromatids se!arate.

/0 phase./a! !hase 1 !rior to DNA s#ntesis/B 1= hrs. ach

chromosome is still a one doule heli,.

S phase.S#nthesis of DNA re!lication/B *hrs

/1 phase./a! !hase 2 et3een DNA s#nthesis and mitosis/B 4 hrs.

ach chromosome is 2 identical chromatids.

/2.cells 3hich hae sto!!ed diiding

*. Dra3 a diagram sho3ing ho3 the chromosomes segregate at meta!hase.


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Nucleic Acids 3 (ene )rganisation and Transcri"tion 1Anil Cho!ra

1. Descrie the asic differences et3een DNA and NA.

DNA is hereditar# and is s!lit u! into functional units called &enes$

&enes code for !roduction of functional NA8s and !roteins 3hich gie cells their

!articular characteristics e.g.o red lood cells !roduce haemogloin

o ?-cell l#m!hoc#tes !roduce antiodies.

(he DNA se>uence of an organism is its &enome$

0uman genome contains et3een 25 === and %5 === genes and this is contained in

: C. 4+ chromosomes" 22 !airs of autosomal chromosomes and one !air of se,

chromosomes E F /.

uired for s!ecific cell function.


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5. Descrie 3hat is meant # a J(ranscri!tion factorJ.

A transcription factorregulates the leel of transcri!tion of a gien gene. )t is also kno3n as

a DNA binding protein. (here are 2 t#!es

(ranscri!tional actiators actiate gene e,!ression.

(ranscri!tional re!ressors su!!ress gene e,!ression.

(hese act together to ring aout changes in e,!ression of genes.,ternal signals such as

tem!erature" hormones" gro3th factors" oltage e.t.c. all hae an effect on transcri!tion

factors.

Mutations in transcri!tion factors cause here%itary %isor%ers" and anormalities in

transcri!tion factor e,!ression are found in man# cancers.

+. Descrie 3ith the aid of diagrams the !rocesses inoled in transcriing a eukar#oticgene.

(he (A(A se>uence in DNA is the !oint at 3hich transcri!tion starts. (his is done # NA

!ol#merase )).

1. (K ))Dcontaining a (A(A inding !rotein/ 3hich !artiall# un3inds the heli,2. (K ))Aand (K ))4ind to the (K ))D.%. NA !ol#merase )) inds to (K ))44. (K ))Fis alread# ound to NA !ol#merase and is @oined # (K ))5" (K ))H" and (K ))65. (his further un3inds the heli, to facilitate the transcri!tion # NA !ol#merase )).


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(ranscri!tion factors increase the leel of transcri!tion on inding # ending7 the DNA and

modif#ing chromatin # ac#lation of histones. (he# can e used in thera!eutic treatment e.g.

Aspirin. As!irin sto!s the reakdo3n of a transcri!tion factor 3hich increases the

!roduction of c#tokines. (his reduces inflammation.


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0alf of all lympho-lastic le+'aemiashae mutated transcri!tion factors.

4reast cancer. reast cancer cells oer e,!ress the transcri!tion factor that is the

oestrogen rece!tor.


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Nucleic Acids * (ene )rganisation and Transcri"tion ++Anil Cho!ra

1. Descrie" 3ith the aid of diagrams" the eents that take !lace in !re- mNA!rocessing.

2. Define 3hat is meant # a s!lice donor site7%. Define 3hat is meant # a s!lice acce!tor site74. Descrie the lariat7 intermediate in mNA s!licing5. Define the function of the 9!liceosome7+. Descrie the addition of a ca!7 and !ol# A tail7 to !re-messenger hn-/ NA.uence /U this is thesplice %onor site/and end 3ith the se>uence A/ this is the splice acceptor site/.

2/ Processing re>uires small rionuclear !roteins. U0inds to the donor site.


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%/ U13 U9and U:all ind to the intron itself" and U!inds to the s!lice acce!tor site.

4/ 9!lice donor site se>uence is cleaed off and the &7 ase from the L& se>uence atthe s!lice donor site cures round to form a !hos!hodiester ond et3een itself andan A7 residue in the intron.

5/ (he !hos!hodiester ond at the &7 end of the intron at the s!ice acce!tor site iscleaed forming a lariat str+ct+re$

+/ A ligase en6#me attaches all the e,ons together.$/ A CAPis attached to the end of the mNA./ A !ol# A tail is added to the end of the mNA one ase at a time. )t is added

do3nstream of the se>uence AALAAA.

$.


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Nucleic Acids 5 ,rotein translation and translationalmodi-ication

Anil 5hopra

1. /utline the mechanisms b which ribosomes can translate an mRNA se2uence into aprotein se2uence.

Brom the mRNA produced in transcription and processing "so onl the exons are in the

final mRNA#, eer base pairs codes for 1 amino acid.

!his is known as a codon.

!here are ;6 codons that code for (: different amino acids, therefore some codons code

for the same amino acid, some are !AR! 5/D/N "+et AC4# and some are !/P

5/D/N.

Rare amino acids hae few codons.

mRNA consists of the !MeG APat one end, and a poly A tail at the other. n between

these there is an untranslated regionat both the & and the %& end.

56ca, 56UTR co$in' re'ion 36UTR ,olyA

78e% AAAAAn


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(. Describe the role of aminoacl tRNAs in ensuring the fidelit of the genetic code.!he processed mRNA leaes the nucleus and attaches to

ribosomes where translation occurs. 0ere tRNA "transfer

RNA# molecules transport amino acids to the ribosome$

!here is onl one tRNA molecule per amino acid and the arecoalentl linked b aminoacyl tRNA synthetases using A!P.

n order to ensure fidelit of the genetic code there are (: tpes of

aminoacl tRNA snthetases so that each amino

acid links with its specific tRNA molecule.

At one end of the tRNA molecule, there is an

anticodon containing the complementar base

pairs which forms an antiparallel relationship with the mRNA.

. tate how a ribosome recognises the start and end of a

se2uence to be translated.Translation:

1# Ribosomes consist of ( subunits, 6: and ;:.

!hese subunits dissociate8split.

(# Preinitiation complex is formed which contains +et'tRNAeBs6: subunit. "+et

is alwas the first amino acid#

# !his then binds to the mRNA.


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6# !

h

e

larger ;: subunit attaches to the mRNA in the same wa. "with the tRNA and eBs

attached#5/ 4!P4DP ensures correct base pairing.

;# !he second tRNA molecule holding the next amino acid in the chin binds to the

mRNA molecule immediatel ad-acent to the preious one i.e. in frame"

9# !he two amino acids are

-oined together with a

peptide bond peptidl

transferase on the ;:

subunit.

E# !he first of tRNA

molecule dissociates

and the second

tRNA is now in the peptidl "P# site.

=# #longation $actors"


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1(# !he ribosome then releases from the mRNA.

6.


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Nucleic Acids . Anal&sis o- DNAAnil Cho!ra

1. ,!lain the term h#ridisation" used for inding of a !roe to a nucleic acid.2. Descrie the reactions carried out # restriction en6#mes restriction

endonucleases/ and e,!lain their usefulness in anal#sis of DNA.DNA Clonin&. is a method of selectiel# am!lif#ing DNA se>uences of interest to generate

homogenous DNA !o!ulations. (here are 2 t#!es

Cell-ased DNA cloning in vivo) Cell-free DNA cloning in vitro)- Pol#merise Chain eaction

Cell ?ased DNA Cloning

1/ (he tar&et DNAand a replicone.g. !lasmid" #east artificialchromosome/ are cut # restriction en%on+cleases$

estriction endonucleases t#!e ))/ 3ork # cleaing DNA at

s!ecific se>uences.

(hese are usuall# short and !allendromic the same ack3ard as

the# are for3ard/.

Can !roduce lunt ends7 or stick#

ends7.

&enerall#" the longer the se>uence cut" the less fre>uentl# it

a!!ears in the DNA.2/ (he DNA fragments are mi,ed and @oined # a DNA li&ase$

%/ (he ne3l# formed recom-inant DNA is introduced into acteria#eastcells.

4/ (he re!licon contains an antiiotic resistance marker therefore onl# those3ith the re!licon to surie 3hen treated 3ith antiiotics.


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5/ (he cell culture is e,!anded and the recominant DNA is isolated.

%. Descrie ho3 DNA fragments can e se!arated on the asis of si6e.

9e!aration of DNA fragments # lectro!horesis

As DNA is a negatiel# charged molecule" it 3ill moe to3ards the !ositie anode if

it is in an electrol#te mi,ture. 9maller fragments of DNA 3ill moe faster than larger

ones and so it is eas# to isolate DNA 3hich has een modified or added to. (his

!rocess is called electrophoresis$ (his can then go on to e hybridised.

0#ridisation

)f one 3ants to find out 3here a s!ecific DNA se>uence is in a chromosome or on a gene" a

hy-ri%isation assayis done

1/ A laelled nucleic acid pro-eis mi,ed in amongst unlaelled nucleotides DNA" NA or

oligonucleotides/. Proes can e made in arious 3a#sa. DNA Proes can either e s#nthesised #

i. Nic' translation " this is 3here a chunk of DNA isremoed nicked7/ first # DNase and the rest #e,onuclease. (hen a certain DNA !ol#merase s#nthesises

that radioactiel# lael the ne3 strand. (his ecomes the !roe.

ii. Ran%om prime% la-ellin&3this is 3here the DNA is denatured andrandom oligonucleotides are added. (he DNA !ol#merase uses theoligonucleotides as !rimers to s#nthesise the ne3 radioactiel# laelled !roe.


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. NA !roes are made # using a radioactiel# laelled !roe in DNA !lasmidsand then adding NA !ol#merase" 3hich !roduced radioactiel# laelled NA!roes.

c. 'ligonucleotides are laelled 3ith a radioactie A(P molecule 3hich is added tothe 58 # an en6#me called Polyn+cleoti%e 'inase$

2/ ?oth

the target DNA and the laelled DNA !roes are denatured either # heating or mi,ing3ith some solution/ and ound to a solid su!!ort e.g. n#lon or nitrocellulose memrane.(his readil# inds single-stranded nucleic acid e.g. denatured DNA/ and thenh#ridised 3ith a solution of radioactiel# or fluorescentl#/ laelled / !roe.

%/ (he# then h#ridise in that some of the laelled !rimers mi, in to the target DNA. (hesecan e recognised # !hotogra!hic film and therefore isolated.

4. ,!lain the conce!t of stringenc# of h#ridisation" and the factors that contriute to

stringenc#.

(he DNA is denatured # heating until all the h#drogen onds

holding the t3o chains together are roken. (his can de!end on the length of the strandlonger chainOmore onds/" the nature of the onds &-C has one more ond than A-(/" andenironment certain atoms such as Na stailise the onds and certain molecules such asurea destailise them/.

(he melting tem!erature is taken as the mid !oint of the transition

et3een doule and single stranded DNA. Around $' C/


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(his is then allo3ed to cool do3n at around 25oC 3here

h#ridisation occurs. i.e. annealing of the DNA strands/

Hy-ri%isation strin&ency i.e. the !o3er to distinguish et3een related se>uences/

increases 3ith

- )ncrease in tem!erature

- Decrease in Na concentration

5. ,!lain ho3 the !ol#merase chain reaction PC/ is used to am!lif# small amounts of

DNA for suse>uent anal#sis.

Pol#merase Chain eaction

(his is a !rocess # 3hich s!ecific target DNA can e am!lified co!ied man# times/ in a

collection of DNA se>uences.

2 !rimers are made one for each strand that is to e co!ied.

(he DNA is heated till it is denatured and so it is sin&le

stran%e%$

A s!ecial en6#me kno3n asThermosta-le Thermophilus

aquaticusDNA polymerase along 3ithdN(Ps e,tend 58-%8 from the !rimers andgenerate ne3 strands.

Denat+re ;9oC

Anneal !27:2oC

5*ten% uence.

(he# are usuall# aout 12 n+cleoti%es in

len&th$

(he# should not inole too man#

re!eated nucleotides as these form hair!ins.

(he !ercentage of C-& onds along 3ith their length should gie

#ou a rough estimate of their melting tem!erature.

Com!lementar# ase !airs at either %8 ends should e aoided in

order to !reent !rimer dimers.


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$. Descrie the !rinci!les ehind the standard method of otaining the se>uence of a !iece

of DNA.

1/ 'tain the tem!late strand #ou 3ish to se>uence" and then add a DNA !rimer

com!lementar# to the %8 end.

2/ .

(hese come 3ith 4 different ases attached to them and therefore 3ill halt the reaction

onl# after that corres!onding ase ho3eer man# times that ma# come u!. Add each one to one reaction.

ou 3ill then" in each reaction" get DNA chains of ar#ing lengths. e.g. in the &7 reaction

3here #ou added dd/(P/" #ou ma# getQo dd/A(&C&

o dd/C&(AA(&C&

o dd/C(&A(C&C&(AA(&C&

4/ (his halts the !roduction of the fluorescentl# marked strands at different !oints.5/ (herefore the strands are all different lengths" 3hich means that in electro!horesis" the#

3ill all a!!ear at different leels.+/ (he mi,tures of the four different reactions are sim!l# !ut in 4 columns 3hich can e

read out from to! to ottom to gie #ou the se>uence this can e done manuall# if thenucleotides hae een radioactiel# marked" or automaticall# if the# hae eenfluorescentl# marked.

Anil Nucleic Acids

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