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I Recombinant DNA Technology

This document provides an overview of several important techniques in molecular biology and genetics, including restriction enzymes, nucleic acid hybridization, DNA cloning, viruses, DNA sequencing, and polymerase chain reaction (PCR). It describes the basic principles and applications of these techniques, such as how restriction enzymes cut DNA at specific sites, how nucleic acid hybridization allows detection of specific sequences, the basic process of DNA cloning including creating DNA libraries, and how PCR amplifies a specific DNA sequence through repeated cycles of heating and cooling of the DNA template.

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Pratik Kulkarni
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43 views37 pages

I Recombinant DNA Technology

This document provides an overview of several important techniques in molecular biology and genetics, including restriction enzymes, nucleic acid hybridization, DNA cloning, viruses, DNA sequencing, and polymerase chain reaction (PCR). It describes the basic principles and applications of these techniques, such as how restriction enzymes cut DNA at specific sites, how nucleic acid hybridization allows detection of specific sequences, the basic process of DNA cloning including creating DNA libraries, and how PCR amplifies a specific DNA sequence through repeated cycles of heating and cooling of the DNA template.

Uploaded by

Pratik Kulkarni
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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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 -

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