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%
Nucleic Acids 1 Nucleic Acids and Chromosomes
Alex Perkins
1. Draw the structure of a nucleotide labelling the sugar, base and phosphates and
explain 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
O
1
2
3
4
5
2
N
N
N
N
NH
H
H
P O
O
O
P O
O
O
P O
O
O
_
_
sugar deo,#ri-ose in DNA
- ri-ose in NA
-ase !hos!hate
Cytosine
(C)
Uracil
(U)
RNA only
Thyine
(T)
!NA only
N
N
O
O
H
H
H
N
N
O
O
CH
3
H
H
N
N
NH
2
O H
H
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 groo.es, and the directionalit of the
chains.
DNA is 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. 1t is not smmetrical.
Adenine
.A/
&uanine
.&/
N
N
N
N
N0
2
0
0
N
N
N
'
0
2
N
0
minor groove
major groove
The DNA double helix
"
O
O
H
H
H
N
H
N
N
N
O
N
2
N
O
O
O
P O O
P O O
O
P O O
O
N
N
N
N
NH
2
O
H
O
N
NH
2
O
5#
3#
3#
3#
5#
5#
OH
"
"
5' end
3' end
A single DNA chain
!he primar se2uence is the linear se2uence of the bases. 3 con.ention, the nucleotide
se2uence is specified in the %& to ,& direction.
!he secondar structure of 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
negati.e 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$s
ore sta(le
2 hy$ro'en (on$s
less sta(le
WatsonCric! base "airs
N
N
N
O
H
H
N
N
N
N
O
N
H
H
H
N
N
N
N
N
N
N
O
O
CH
3
H
H
H
Melting and re-annealing
0igh temperature and8or low salt concentration causes the two strands to melt or
disassociate.
1f ou then lower the temperature or increase the salt concentration, the two melted
strands will re'anneal into a double helix.
0bridisation$ in a mixture of DNA with different se2uences, the complementar strands
will find each other in the mixture.
%. 5ompare the genomes of E.coli and Homo 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 <.coli DNA is 1.6 mm.
!he DNA in E.coli is tightl packaged the bacterium is onl , m long.
The human genome
!he human genome "diploid# consists of about ; x 1:
=
base pairs of DNA.
!he DNA is di.ided into chromosomes that each contain a linear double'helical DNA
molecule of about (:: x 1:
;
base pairs.
Prior to cell di.ision, the DNA condenses into discrete chromosomes, .isible b
microscop.
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 le.el 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
A(o&t 2++ (ase ,airs o- !NA .ra,,e$ aro&n$
the histones (core !NA)
Histone 1 (et.een the
n&cleosoes
/ histones0 2 each o- 2A1 221 3 an$ 4
A nucleosome
3hort re'ion o- !NA $o&(le heli4
2ea$s on a strin' -or o- chroatin
3+ n chroatin -i(er o- ,ac*e$
n&cleosoes
3ection 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
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 @#
' Aex chromosomes$ ?? for female* ?@ for male
G-banded karyotype of a Normal Male ell
!wo homologues of each chromosome
Nucleic Acids # DNA $e"lication% the Cell C&cle and
'itosis
Anil Cho!ra
1. 1,!lain semi-conser2ati2e re!lication.
Ne3 co!ies of DNA made should -e identical.
1ach daughter cell inherits one older strand and one ne3 strand.
1ach strand ser2es as a tem!late.
2. Descri-e the reaction catal#sed -# DNA !ol#merases.
DNA Helicase4 uses A(P to -reak the h#drogen -onds -et3een the -ase
!airs to 5un6i!7 the DNA.
DNA Polymerase4 s#nthesises ne3 DNA -# adding nucleotides to the
%8 .!rime/ end of the gro3ing chain.
%. Descri-e ho3 nucleoside analogs can -e used as drugs.
9ome nucleoside analogs can -e used as drugs -ecause the#
terminate chains. 1.g.
Ac#clo2ir used in treatment for 0er!es
C#tosine ara-inose used in chemothera!#
Dideo,#c#tosine .ddC/ 0): drug
A6idoth#midine .A;(/ 0): drug.
4. Descri-e the functions of the com!onents of the re!lication com!le, in !rokar#otes
including the terms tem!late" !rimer" leading strand" lagging strand" 'kasaki fragment
and re!lication fork.
DNA !ol#merases need a template and a primer.
(he re!lication !rocess -egins at discrete !oints and occurs -i-directionall#. IT
ALWAYS OCCURS FRO !" TO #"$
'n the lea%in& stran% continuous s#nthesis
'n the la&&in& stran% discontinuous s#nthesis
1. DNA helicase un3inds heli,.
2. <hen a-out 1=== -ases are e,!osed" a short NA !rimer of a-out 5-1= -ases is
s#nthesised -# primase .an NA !ol#merase/.
%. DNA !ol#merase ))) continuall# adds deo,#nucleotide
!hos!hates onto the end of the !rimer to form the chain
e,tension. (his re>uires A(P.
4. 'n the lagging strand there are man# NA !rimers that
are added to 2arious !oints on the tem!late strand.
(heses are acted on -# DNA !ol#merase ))) to !roduce
O'a(a'i )ra&ments. (his also re>uires A(P. (he
lagging strand loo!s around so that the DNA
!ol#merase ))) can 3ork on -oth strands at the
same time.
5. <hen the 'ka6aki fragment reaches the NA
!rimer in front of it" DNA !ol#merase ) remo2es
the NA !rimer using a 58 to %8 e,onuclease.
(he DNA !ol#merase ) s#nthesi6es the ne3
DNA through the NA !rimer region.
+. ?inding !roteins -ind to the lagging strand in order to !re2ent local secondar#
structures forming.
$. (he 'ka6aki fragments are then @oined together -# DNA li&ase$
5. D
e
s
c
r
i
-
e
ho3 accurac# is maintained
-# !roof-reading
and the use of NA
!rimers.
(he high
fidelit# of
DNA re!lication re>uires a !roof-reading
mechanism to ensure no mistakes are made.
Mutations .changes in DNA se>uence/ are 2er# dangerous to the organism. An# errors in
re!lication cannot -e re!aired.
DNA re!lication has an error fre>uenc# of a-out 1 change !er 1=
*
-ase !airs.
?efore a ne3 nucleotide is added" the !re2ious 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 then
h#drol#sed -# the e*on+clease acti,ity of DNA
!ol#merase ))) and the correct -ase is added.
)n errors 3ith NA !rimers" the NA !rimer is
sim!l# remo2ed and re!laced 3ith accurate DNA
s#nthesis.
+. Dra3 a diagram sho3ing re!lication of the 1.coli
chromosome.
(he 1.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 1.coli
chromosome uses different en6#mes.
$. Descri-e the re!lication of mammalian
chromosomes.
Mammalian chromosomes are long and linear.
e!lication originates at 2arious different !oints along
the DNA at inter2als of around 1==kilo-ase !airs and !roceeds -i-directionall#. )t finishes 3hen all the
forks ha2e met.
(here are at least 5 different DNA !ol#merases4
Pol a s#nthesises the !rimers for -oth leading and lagging strands
Pol - is e>ui2alent to Pol ) in 1.coli
Pol % s#nthesises the leading strand
Pol e s#nthesises the lagging strand
Pol & re!licates mitochondrial DNA
A. Descri-e the different !hases of the cell
c#cle.
phase. itosisB cell di2isionB 1hr. (his is 3here the 2
chromatids se!arate.
/0 phase. /a! !hase 1 .!rior to DNA s#ntesis/B 1= hrs. 1ach
chromosome is still a one dou-le heli,.
S phase. S#nthesis of DNA .re!lication/B *hrs
/1 phase. /a! !hase 2 .-et3een DNA s#nthesis and mitosis/B 4 hrs.
1ach chromosome is 2 identical chromatids.
/2. cells 3hich ha2e sto!!ed di2iding
*. Dra3 a diagram sho3ing ho3 the chromosomes segregate at meta!hase.
Nucleic Acids 3 (ene )rganisation and Transcri"tion 1
Anil Cho!ra
1. Descri-e the -asic differences -et3een DNA and NA.
DNA is hereditar# and is s!lit u! into functional units called &enes$
&enes code for !roduction of functional NA8s and !roteins 3hich gi2e cells their
!articular characteristics e.g.
o red -lood cells !roduce haemoglo-in
o ?-cell l#m!hoc#tes !roduce anti-odies.
(he DNA se>uence of an organism is its &enome$
0uman genome contains -et3een 25 === and %5 === genes and this is contained in
1:1C C1DD.
4+ chromosomes" .22 !airs of autosomal chromosomes and one !air of se,
chromosomes E F C/.
<hen genes are e*presse%3 the# are used in that !articular cell. 9ome Ghousekee!ing
genes8 are e,!ressed in all cells. 25H of genes are re>uired for s!ecific cell function.
<hen genes are e,!ressed the# !roduce" NA .ri-onucleic acid/. (his is the same as
DNA e,ce!t for that4
o )t is sin&le stran%e%$
o (he -ase th#mine is re!laced 3ith Uralic.
o (he !entose sugar is ri-ose. .not deo,#ri-ose/
o (here are % different t#!es of it4 mNA" tNA and rNA.
2. Descri-e 3hat is meant -# Itranscri!tionI.
)n the !rocess of transcri!tion" NA is !roduced as a tem!late for !rotein translation.
(his al3a#s occurs in the nucleus.
%. Dist the ma@or functional classes of NA and the classes of NA !ol#merase in2ol2ed
in s#nthesising each of these.
&ene transcri!tion is carried out -# en6#mes called 5NA Pol#merases7. (here are %
t#!es
o NA Pol#merase ) -(ranscri-es rNA genes.
o NA Pol#merase )) - (ranscri-es genes encoding !roteins into mNA.
o NA Pol#merase )))- (ranscri-es tNA and 59 NA genes.
4. Descri-e 3hat is meant -# a Jgene !romoterJ.
A gene promoter is a DNA se>uence at 3hich the transcri!tion initiation com!le,
assem-les. (his com!le, is needed for the elongation reaction of NA s#nthesis to occur.
5. Descri-e 3hat is meant -# a J(ranscri!tion factorJ.
A transcription factor regulates the le2el of transcri!tion of a gi2en gene. )t is also kno3n as
a DNA binding protein. (here are 2 t#!es4
(ranscri!tional acti2ators acti2ate gene e,!ression.
(ranscri!tional re!ressors su!!ress gene e,!ression.
(hese act together to -ring a-out changes in e,!ression of genes. 1,ternal signals such as
tem!erature" hormones" gro3th factors" 2oltage e.t.c. all ha2e an effect on transcri!tion
factors.
Mutations in transcri!tion factors cause here%itary %isor%ers" and a-normalities in
transcri!tion factor e,!ression are found in man# cancers.
+. Descri-e 3ith the aid of diagrams the !rocesses in2ol2ed in transcri-ing a eukar#otic
gene.
(he (A(A se>uence in DNA is the !oint at 3hich transcri!tion starts. (his is done -# NA
!ol#merase )).
1. (K ))D .containing a (A(A -inding !rotein/ 3hich !artiall# un3inds the heli,
2. (K ))A and (K ))4 -ind to the (K ))D.
%. NA !ol#merase )) -inds to (K ))4
4. (K ))F is alread# -ound to NA !ol#merase and is @oined -# (K ))5" (K ))H" and (K ))6
5. (his further un3inds the heli, to facilitate the transcri!tion -# NA !ol#merase )).

(ranscri!tion factors increase the le2el of transcri!tion on -inding -# 5-ending7 the DNA and
modif#ing chromatin -# ac#lation of histones. (he# can -e used in thera!eutic treatment4 e.g.
Aspirin. As!irin sto!s the -reakdo3n of a transcri!tion factor 3hich increases the
!roduction of c#tokines. (his reduces inflammation.
0alf of all lympho-lastic le+'aemias ha2e mutated transcri!tion factors.
4reast cancer. -reast cancer cells o2er e,!ress the transcri!tion factor that is the
oestrogen rece!tor.
Nucleic Acids * (ene )rganisation and Transcri"tion ++
Anil Cho!ra
1. Descri-e" 3ith the aid of diagrams" the e2ents that take !lace in !re- mNA
!rocessing.
2. Define 3hat is meant -# a 5s!lice donor site7
%. Define 3hat is meant -# a 5s!lice acce!tor site7
4. Descri-e the 5lariat7 intermediate in mNA s!licing
5. Define the function of the 59!liceosome7
+. Descri-e the addition of a 5ca!7 and 5!ol# A tail7 to !re-messenger .hn-/ NA.
<hen NA is !roduced initiall# from the gene is still not read# to -e used in translation #et. )t
is kno3n as Pre7mRNA3 primary transcript or hetero&eno+s n+clear hn RNA.
)t needs to -e !rocessed first. (his occurs in the nucleus.
DNA is formed from
o 5*ons 8 !arts of the DNA that 3ill -e transcri-ed in -oth the !re-mNA and the
final mNA.
o Introns 8 !arts of the DNA that 3ill -e transcri-ed in the !re-mNA -ut then
edited out in the final mNA.
(his occurs in a series of ste!s4
1/ (he introns that are to -e edited out 3ill start 3ith the se>uence /U .this is the
splice %onor site/ and end 3ith the se>uence A/ .this is the splice acceptor site/.
2/ Processing re>uires small ri-onuclear !roteins. U0 -inds to the donor site.
%/ U13 U9 and 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 clea2ed off and the 5&7 -ase from the L& se>uence at
the s!lice donor site cur2es round to form a !hos!hodiester -ond -et3een itself and
an 5A7 residue in the intron.
5/ (he !hos!hodiester -ond at the 5&7 end of the intron at the s!ice acce!tor site is
clea2ed forming a lariat str+ct+re$
+/ A ligase en6#me attaches all the e,ons together.
$/ A CAP is attached to the end of the mNA.
A/ 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.
$. <ith e,am!les" descri-e ho3 mutations in s!lice sites feature in human disease.
%%H of mutations in inherited diseases occur at s!lice donorMacce!tor sites.
Thalassaemia. this is a disorder in 3hich there is an im-alance in the relati2e amounts of
glo-ulin chains in red -lood cells. -thalassaemia is 3hen there is a deficienc# of chains.
(his results in se2er anaemia" 3hich then results in increase in iron u!take causing
hepatome&aly .enlargement of the li2er/" %ar'enin& o) s'in .due to iron-stimulated melanin
!roduction/" car%iomyopathy.
Nucleic Acids 5 ,rotein translation and translational
modi-ication
Anil 5hopra
1. /utline the mechanisms b which ribosomes can translate an mRNA se2uence into a
protein se2uence.
Brom the mRNA produced in transcription and processing "so onl the exons are in the
final mRNA#, e.er , base pairs codes for 1 amino acid.
!his is known as a codon.
!here are ;6 codons that code for (: different amino acids, therefore some codons code
for the same amino acid, some are A!AR! 5/D/NA "+et AC4# and some are A!/P
5/D/NA.
Rare amino acids ha.e few codons.
mRNA consists of the !MeG AP at one end, and a poly A tail at the other. 1n between
these there is an untranslated region at both the ,& and the %& end.
56ca, 56UTR co$in' re'ion 36UTR ,olyA
78e%
AAAAA
n
(. Describe the role of aminoacl tRNAs in ensuring the fidelit of the genetic code.
!he processed mRNA lea.es 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 are
co.alentl linked b aminoacyl tRNA synthetases using A!P.
1n 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.
,. Atate how a ribosome recognises the start and end of a
se2uence to be translated.
Translation:
1# Ribosomes consist of ( subunits, 6:A and ;:A.
!hese subunits dissociate8split.
(# Preinitiation complex is formed which contains +et'tRNADe1BsD6:A subunit. "+et
is alwas the first amino acid#
,# !his then binds to the mRNA.
6# !
h
e
larger ;:A subunit attaches to the mRNA in the same wa. "with the tRNA and e1Bs
attached#
5/ 4!P 4DP 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 pre.ious one i.e. in frame"
9# !he two amino acids are
-oined together with a
peptide bond peptidl
transferase on the ;:A
subunit.
E# !he first of tRNA
molecule dissociates
and the second
tRNA is now in the peptidl "P# site.
=# #longation $actors "<Bs# use the energ of 4!P to enhance the efficienc and
accurac of translation b pro.iding FpausesG "e.g. 4!P hdrolsis# that allow
incorrect base pairs to dissociate.
1:# !his continues until it reaches the stop codon"
11# 7hen this is recognised, release factors bind to the empt A site and the peptide
chain is released.
1(# !he ribosome then releases from the mRNA.
6. <xplain wh some antibiotics inhibit protein snthesis in prokarotes but not
eukarotes.
Antibiotics are used to inhibit the action of prokarotes. !he do this b interfering with
stages of their replication or protein snthesis as both of these are complex procedures
re2uiring man steps. <.g.$
H Atreptomcin 1nhibits initiation
H !etraccline 1nhibits tRNA binding
H <rthromcin 1nhibits translocation
H 5hloramphenicol 1nhibits peptide transfer
H Puromcin !erminates elongation
H 5cloheximide 1nhibits peptide transfer "in eukarotes#
%. 1dentif the features of a newl'snthesised protein that are re2uired for it to enter the
secretor pathwa.
As cells contain man compartments, there needs a mechanism to transfer proteins across
membranes$
!he first (:'(6 amino acids are a signal se%uence"
!his is recognised and bound to b a signal recognition particle &RP"
!his ARP binds to receptors on the surface of the <R "or other compartment#.
!he amino acid chain is then fed through to the R<R lumen where the signal se2uence is
clea.ed of and the amino acid chain is folded.
;. 4i.e examples of the was in which newl'snthesised proteins can be post'
translationall modified.
After snthesis, the proteins undergo post-translational modification which
in.ol.es .arious things including$
H Proteoltic clea.age "e.g. insulin 'I A and 3 chains#
H Disulphide bond formation "e.g. insulin#
H Addition of carbohdrate "4lcoslation#
H Addition of phosphate
"Phosphorlation#
H Addition of lipid groups
"Prenlation, Aclation#
Nucleic Acids . Anal&sis o- DNA
Anil Cho!ra
1. 1,!lain the term h#-ridisation" used for -inding of a !ro-e to a nucleic acid.
2. Descri-e 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 selecti2el# am!lif#ing DNA se>uences of interest to generate
homogenous DNA !o!ulations. (here are 2 t#!es4
Cell--ased DNA cloning .in vivo)
Cell-free DNA cloning .in vitro) - Pol#merise Chain eaction
Cell ?ased DNA Cloning
1/ (he tar&et DNA and a replicon .e.g. !lasmid" #east artificial
chromosome/ are cut -# restriction en%on+cleases$
estriction endonucleases .t#!e ))/ 3ork -# clea2ing DNA at
s!ecific se>uences.
(hese are usuall# short and !allendromic .the same -ack3ard as
the# are for3ard/.
Can !roduce 5-lunt ends7 or 5stick#
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 -acteriaM#east
cells.
4/ (he re!licon contains an anti-iotic resistance marker therefore onl# those
3ith the re!licon to sur2i2e 3hen treated 3ith anti-iotics.
5/ (he cell culture is e,!anded and the recom-inant DNA is isolated.
%. Descri-e ho3 DNA fragments can -e se!arated on the -asis of si6e.
9e!aration of DNA fragments -# 1lectro!horesis
As DNA is a negati2el# charged molecule" it 3ill mo2e to3ards the !ositi2e anode if
it is in an electrol#te mi,ture. 9maller fragments of DNA 3ill mo2e 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 assay is done4
1/ A la-elled nucleic acid pro-e is mi,ed in amongst unla-elled nucleotides .DNA" NA or
oligonucleotides/. Pro-es can -e made in 2arious 3a#s
a. DNA Pro-es can either -e s#nthesised -#4
i. Nic' translation " this is 3here a chunk of DNA is
remo2ed .5nicked7/ first -# DNase and the rest -#
e,onuclease. (hen a certain DNA !ol#merase s#nthesises
that radioacti2el# la-el the ne3 strand. (his -ecomes the !ro-e.
ii. Ran%om prime% la-ellin&3 this is 3here the DNA is denatured and
random oligonucleotides are added. (he DNA !ol#merase uses the
oligonucleotides as !rimers to s#nthesise the ne3 radioacti2el# la-elled !ro-e.
-. NA !ro-es are made -# using a radioacti2el# la-elled !ro-e in DNA !lasmids
and then adding NA !ol#merase" 3hich !roduced radioacti2el# la-elled NA
!ro-es.
c. 'ligonucleotides are la-elled 3ith a radioacti2e A(P molecule 3hich is added to
the 58 -# an en6#me called Polyn+cleoti%e 'inase$
2/ ?
o
t
h
the target DNA and the la-elled DNA !ro-es are denatured .either -# heating or mi,ing
3ith some solution/ and -ound to a solid su!!ort e.g. n#lon or nitrocellulose mem-rane.
(his readil# -inds single-stranded nucleic acid .e.g. denatured DNA/ and then
h#-ridised 3ith a solution of .radioacti2el# or fluorescentl#/ la-elled .N/ !ro-e.
%/ (he# then h#-ridise in that some of the la-elled !rimers mi, in to the target DNA. (hese
can -e recognised -# !hotogra!hic film and therefore isolated.
4. 1,!lain the conce!t of stringenc# of h#-ridisation" and the factors that contri-ute 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 strand
.longer chainOmore -onds/" the nature of the -onds .&-C has one more -ond than A-(/" and
en2ironment .certain atoms such as Na
P
sta-ilise the -onds and certain molecules such as
urea desta-ilise them/.
(he melting tem!erature is taken as the mid !oint of the transition
-et3een dou-le and single stranded DNA. .Around A$
'
C/
(his is then allo3ed to cool do3n at around 25
o
C 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 3ith4
- )ncrease in tem!erature
- Decrease in NaP concentration
5. 1,!lain ho3 the !ol#merase chain reaction .PC/ is used to am!lif# small amounts of
DNA for su-se>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 as Thermosta-le Thermophilus
aquaticus DNA polymerase along 3ith
dN(Ps e,tend 58-%8 from the !rimers and
generate ne3 strands.
Denat+re ;9
o
C
Anneal !27:2
o
C
5*ten% <1
o
C
(his c#cle is re!eated
man# times.
+. Descri-e in general terms the 3a# in 3hich PC
!rimers 3ould -e selected to am!lif# a gi2en DNA
se>uence.
(he# are usuall# a-out 12 n+cleoti%es in
len&th$
(he# should not in2ol2e too man#
re!eated nucleotides as these form hair!ins.
(he !ercentage of C-& -onds along 3ith their length should gi2e
#ou a rough estimate of their melting tem!erature.
Com!lementar# -ase !airs at either %8 ends should -e a2oided in
order to !re2ent !rimer dimers.
$. Descri-e the !rinci!les -ehind the standard method of o-taining the se>uence of a !iece
of DNA.
1/ '-tain the tem!late strand #ou 3ish to se>uence" and then add a DNA !rimer
com!lementar# to the %8 end.
2/ <ith DNA !ol#merase" s#nthesise the com!lementar# strand.
%/ (hen use this ne3l# formed com!lementar# strand to s#nthesise the original DNA using
fluorescent or radioacti2el# marked nucleotides in 9 %i))erent reactions$ )n each of these
reactions" add a small amount of %%NTPs =%i%eo*yri-on+cleosi%e triphosphate>.
(hese come 3ith 4 different -ases attached to them and therefore 3ill halt the reaction
onl# after that corres!onding -ase ho3e2er man# times that ma# come u!.
Add each one to one reaction.
Cou 3ill then" in each reaction" get DNA chains of 2ar#ing lengths. e.g. in the 5&7 reaction
.3here #ou added dd/(P/" #ou ma# getQ
o 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 le2els.
+/ (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 gi2e #ou the se>uence this can -e done manuall# if the
nucleotides ha2e -een radioacti2el# marked" or automaticall# if the# ha2e -een
fluorescentl# marked.