1 Restriction enzymes

2 Nucleic acid hybridization

3 DNA cloning
4 Viruses
5 DNA sequencing
6 olymerase chain reaction
! Recombinant DNA technology

"#er#ie$

Restriction enzyme digestion

Nomenclature

%el electro&horesis

Restriction ma&s

Restriction 'ragment length

&olymor&hisms(R)*+
Restriction enzymes
"#er#ie$

Restriction enzymes allo$ DNA to be

cut at s&eci'ic sites ,Nucleic acid
hybridization allo$s the detection o'
s&eci'ic nucleic acid sequences ,DNA
sequencing can be used to easily
determine the nucleotide sequence o'
a DNA molecule-
Restriction endonuclease
(Restriction enzyme+

Bacterial enzymes which cut DNA into

defined and reproducible fragments

Identified in the late 1960s

ey disco!ery which allowed the DNA

cloning to become a reality
Restriction endonuclease
(origination+

"ne component of the bacterial restriction#modification

system$ a natural defense mechanism of bacteria to against
the introduction of foreign DNA into the cell

%estriction endonuclease& recognize a short$ symmetrical

DNA se'uence$ and cut DNA bac(bone in each strand at a
specific site within that se'uence )(ill foreign DNA*

+ythylase& methylates , or A of the cellular DNA

.y&es o' Restriction
endonuclease
.y&e ! .y&e !! .y&e !!!
)unctions /ndonuclease 0
methylase
/ndonuclease /ndonuclease
1onditions A.2 3b
24
3g
24
A.2 3g
24
Recognition
sequences
/co56 AA1N
6
%.%1
/co76 .%AN
8
.%1.
alindromic(

/co16 A%A11
/co156 1A%1A%
1utting sites At least 1999b& a$ay At or close to recog-
seq
24:26 b& a$ay
Restriction enzymes
Recognize 4-8 bp palindromic sequences. Most
commonly used enzymes recognize 6 bp which
occurs at a rate of 4
6
=4!6 bp. "4
4
=#$6 bp%
4
8
=6$$&6 bp'
(. )ighly specific
#. *ommercially a+ailable
&. Require Mg#, for enzymatic acti+ity
4. *ompatible ends from different enzymes-
5 GAATTC 3
3 CTTAAG 5
e-g- EcoRI site&
Recognition sequences
$. protruding ends &. protruding ends
5-CCCGGG-3
3-GGGCCC-5
5-CCC-OH
3-GGG- p
p -GGG-3
OH-CCC-5
+
SmaI
blunt ends
*ohesi+e/stic0y ends
Restriction sequences
Restriction digestion
1garose2 a polysaccharide deri+ed from seaweed-
which forms a solid gel when dissol+ed in aqueous
solution ".$3-#3'
- ve electrode
+ ve electrode
Negatively charged DNA
Agarose gel electro&horesis
supercoiled
nicked
1garose gel electrophoresis
*o+alently 4oin the 561 molecules
with the base-pairing cohesi+e
ends- or blunt ends- if the $.-ends
ha+e phosphate groups.
561 ligation
.
if the !ector is phosphorylated
Recombinant 561 molecules
)ig
.he hybridization reaction
3onitoring s&eci'ic nucleic acid sequences
;outhern blotting
Northern blotting
!n situ hybridization
Nucleic acid hybridization
.he hybridization reaction

Double:stranded DNA denatures into single

strands as the tem&erature rises but renatures
into a double:stranded structure as the
tem&erature 'alls - Any t$o single:stranded
nucleic acid molecules can 'orm double:
stranded structures (hybridize + &ro#ided that
ha#e su''icient com&lementary nucleotide
sequence to ma<e the resulting hybrid stable
under the reaction conditions -
3onitoring s&eci'ic nucleic acid
sequences

.he concentration o' s&eci'ic nucleic acid

sequence in a sam&le can be measured by
hybridization $ith a suitable labeled DNA &robe -
A'ter hybridization2 nuclear is used to destroy
unhybridized &robe and the &robe remaining is a
measure o' the concentration o' the target
sequence -
;outhern biotting

;outhern blotting in#ol#es electro&horesis o'

DNA molecules in an agarose gel and then
blotting the se&arated DNA bands on to a
nitrocellulose 'ilter -.he 'ilter is then incubated
$ith a labeled DNA &robe to detect those seq
&arated DNA bands that contain sequences
com&lementary to the &robe -
Northern blotting

Northern blotting is analogous to

;outhern blotting e=ce&t that the
sam&le nucleic acid that is se&arated by
gel electro&horesis is RNA rather than
DNA
!n situ hybridization

)or in situ hybridization2 a tissue sam&le is

incubated $ith a labeled nucleic acid &robe2
e=cess &robe is $ashed a$ay and the location o'
hybridized &robe is e=amined- .he technique
enables the s&atial localization o' gene
e=&ression to be determined as $ell as the
location o' indi#idual genes on chromosomes-

.he &rinci&le o' DNA cloning

.he basics o' DNA cloning

DNA libraries

;creening DNA libraries

DNA cloning
7asic &rocedure o' DNA cloning
561 libraries

DNA libraries
DNA libraries are sets of DNA clones, each of which
has been derived from the insertion of a different
fragment into a vector followed by propagation in the
host.
1 clone is a genetically distinct indi+idual or set of
identical indi+iduals
7enomic
561
561 libraries *561 libraries
7enomic libraries
prepared form random fragments of
genomic 561- which may be
inefficient to find a gene because of
the huge abundance of the non-
coding 561
c561 libraries
DNA copies cDNA! synthesized from
the mRNA by reverse transcription are
inserted into a vector to form a cDNA
library. "#ch more efficient in
identifying a gene, b#t do not contain
DNA coding for f#nctional RNA or
noncoding se$#ence.

"#er#ie$

7acterio&hages

Animal #iruses
#irus
"#er#ie$

A #irus &article (#irion+ has a DNA or

RNA genome &ac<aged inside a &rotein
ca&sid-
/ach #irus can re&licate only by in'ecting
a limited range o' host cells- Viruses e=it
the host cell by budding through the
&lasma membrane $ithout causing cell
death-
7acterio&hages

7acterio&hages adsorb to a bacterial cell

sur'ace and in>ect the &hage DNA
through the cell $all into the cytosol- !n
the lytic cycle2 this DNA then re&licates
inside the cell and is &ac<aged $ithin
ne$ly synthesized ca&sids2 e#entually
being released by cell lysis-
Animal #iruses

ermissi#e cells in'ected $ith an animal

DNA #irus enter a lytic cycle2 but in
non&ermissi#e cells an animal #irus may
become integrated into the
nucleargenome or become a &lasmid-!n
this case the #irus is <no$n as a DNA
tumor #irus-

.$o methods 'or DNA sequencing

Chain termination method

Automated DNA sequencing

DNA sequencing
.$o methods 'or DNA sequencing

DNA can be sequenced by the chemical

method or the chain termination
&rocedure- .he latter is no$ the method o'
choice in $hich the (single:stranded+ DNA
to be sequenced ser#es as the tem&late
'or the synthesis o' a com&lementary
strand $hen su&&lied $ith a s&eci'ic
&rimer and /-coli DNA &olymerase -
1hain termination method

)our incubation mi=tures are set

u&2each containing the DNA tem&late2 a
s&eci'ic DNA& &rimer2/-coli DNA
&olymerase and all 'our
deo=yribonucleoside tri&hos&hates
(dN.s+- !n addition2 each mi=ture
contains a di''erent dideo=ynucleoside
tri&hos&hate analog2ddA.2dd1.
dd%. or dd..-
Automated DNA sequencing

Automated DNA sequencing uses the chain

termination method but $ith an
oligonucleotide &rimer labeled $ith a
'luorescent dye- .he order in $hich the
di''erent 'luorescently labeled termination
&roducts elute 'rom the gel gi#es the DNA
sequence

rinci&les o' 1R A&&lications

o' 1R
olymerase 1hainReaction
A&&lications o' 1R

1R has made a huge im&act in

molecular biology2 $ith many
a&&lications in areas such as cloning2
sequencing2 the creation o' s&eci'ic
mutations2 medical diagnosis and
'orensic medicine-
rinci&les o' 1R

.he &olymerase chain reaction(1R+

allo$s an e=tremely large number o'
co&ies to be synthesized o' any gi#en
DNA sequence 2$hich consists o' three
ste&s 6denaturation2 &rimer annealing
and elongation -