Techniques of Molec

ular Biology
 Dissecting
the genome into
manageably-sized
segments for
manipulation and
analysis of
DNA sequences

How
the genetic
processes of cell
work?

Finding Separating
the tools Individual
of macromolecules
genetic analysis from the mixtures
-model organism found in the cell
1. Electrophoresis through a Gel Separates
DNA and RNA Molecules According to Size

 Gel electrophoresis
separates DNA molecu
les according to their
size (including molecu
lar weight, shape, cha
rge, topological prope
rties etc.)
subjected to an elec
tric field through a g
el matrix
 reveal the bands by st
aining the gel with fl
uorescent dyes, such a
s ethidium
Gel matrix

Polyacrylamide
 High resolving power
 Separate DNA only over a narrow size range

Agarose
 Less resolving power
 Separate from one another DNA molecules of
up to tens, and even hundreds, of kilobases
Pulsed-field gel electrophoresis

 Very long DNA mole

cules (eg. entire
bacterial or fungi
chromosomes) can b
e resolved from on
e another with the
electric field app
lied in pulses tha
t are oriented ort
hogonally to each
other.
Electrophoresis of RNA

 Single-stranded RNA molecules bear e

xtensive secondary and tertiary stru
cture, which influences their electr
ophoretic mobility
 Glyoxalated RNAs are unable to form
high order structures and hence migr
ate with a mobility that is approxim
ately proportional to molecular weig
ht.
2.Restriction Endonucleases Cleave DNA
Molecules at Particular Sites
 Restriction Endonucleases
← flush end

← stagger ends

↙
Digestion of a DNA fragment with endon
ucleases EcoRI
Restriction Endonucleases
 The use of multiple enzymes allows differ
ent regions of a DNA molecules to be isol
ated
 It also allows a given molecule to be ide
ntified.
 A given molecule will generate a characte
ristic series of patterns when digested w
ith a set of different enzymes
 Different restriction endonucleases have
different cut frequency
3.DNA Hybridization Can Be Used to Ide
ntify Specific DNA Molecules

 Hybridization
the process of base-pairing betwee
n complementary single-stranded poly
nucleotides from two different sourc
es under the appropriate conditions
of ionic strength and temperature.
Hybridization Probes Can Identify Electrophoretical
ly-separated DNAs and RNAs

 Probe with defined sequence – eithe

r a purified fragment or a chemicall
y synthesized DNA molecule is used t
o search mixtures of nucleic acids f
or molecules containing a complement
ary sequence.
 Probe must be labeled in the first p
lace.
Methods for labeling DNA

 Synthesizing new DNA in the presence

of a labeled precursor modified with
either a fluorescent moiety or radio
active atoms by using PCR or hybridi
zing short random hexameric oligonuc
leotides to DNA and allowing a DNA p
olymerase to extend them.
 Adding a label to the end of an inta
ct DNA molecule
Southern blot hybridization
ETOH ppt
Phenol ProtK SDS
Chloro
Spin
tissue

Paper Towels
Northern blot hybridization

 Hybridizing between complementary st

rands of DNA and RNA
 Identify a particular mRNA in a popu
lation of RNAs
 The protocol is basically the same a
s southern blotting
 Difference is that relatively short
RNAs need not be digested with any e
nzymes
4.Isolation of specific segment of DNA

 Isolation of specific segment of DNA

allows further study of that particu
lar DNA molecule, such as DNA sequen
cing, PCR, DNA cloning (creating rec
ombinant DNA molecules) etc.
 DNA can also be expressed with its p
roduct studied.
5.DNA Cloning
 The ability to construct recombinant DNA
molecules and maintain them in cell.
 Components
Vector
insert DNA
Restriction enzyme
Dna ligase
Host organism
Vector
 Three characteristics
 They contain an origin of replication tha
t allows then to replicate independently
of the chromosome of the host.
 They contain a selectable marker that all
ows cells that contain the vector (and an
y attached DNA) to be readily identified.
 They have single sites for one or more re
striction enzymes, which allows DNA fragm
ents to be inserted at a defined point wi
thin an otherwise intact vector
Vector
 Most common vector
plasmid
 Expression vectors
Vectors not only allow the isolation and
purification of a particular DNA, but als
o drive the expression of genes within th
e insert DNA.
Expression vectors have transcriptional
promoters immediately adjacent to the sit
e of insertion.
Transformation
 Transformation
The process by which a host organism can
take up DNA from its environment.
 Some bacteria naturally have genetic comp
etence (the ability to be transformed).
 Calcium-treated cells are competent to be
transformed.
 transformation is inefficiency.
Cloning in a plasmid vector

 A fragment of DNA, generated

by cleavage with EcoRI, is inse
rted into the plasmid vector lin
earized by that same enzyme.

 Once ligated, the recombinant

plasmid is introduced into bact
eria, by transformation.

 Cells containing the plasmid ca

n be selected by growth on the
antibiotic to which the plasmid
confers resistance.
Libraries of DNA Molecules

 DNA library
a population of identical vectors th
at each contains a different DNA ins
ert
Genomic Library
 Genomic library de
rived from total g
enomic DNA cleaved
with a restriction
enzyme.
 It is useful when
generating DNA for
sequencing a genom
e.
cDNA library
 A cDNA (copy DNAs)
library convert mR
NA into DNA sequen
ce using reverse t
ranscriptase.
 It is useful when
the objective is t
o clone a DNA frag
ment encoding a pa
rticular gene.
Hybridization Can Be Used to Identify a spe
cific Clone in a DNA Library

 colony hybridization
The process by whic
h a labeled DNA probe
is used to screen a l
ibrary
Note: If the library is made
using a phage vector, t
hey can be screened in
much the same way as
plasmid library. The diff
erence is the plaques r
ather than colonies are
screened.
6.Chemically Synthesized Oligonucl
eotides
 The 5’-hydroxyl group is b
locked by the addition of
a dimethoxyltrityl protectin
g group.
 The growth of the DNA ch
ain is by addition to the 5’
end of the molecule.
site-directed mutagenesis

 Short DNA molecules up to 30 bases can be che

mically synthesized efficiently and accurately.

 A custom-designed oligonucleotide can harbor a

mismatch to a segment of cloned DNA.
7.The Polymerase Chain Reaction (P
CR)
8.Nested Sets of DNA Fragments Reveal
Nucleotide Sequencing

 The ultimate in probing a genome with hi

gh selectivity, which permits us to find an
y specific sequence with great rapidity a
nd accuracy through the use of a comput
er and appropriate algorithms.
The underlying principle of DNA
sequencing

 Separation of nested sets (the A,T,C,G set

) of DNA molecules by size
 The different lengths of these fragments ca
n be determined by electrophoresis through
a polyacrylamide gel
 Alternatively, the four nested sets can be
differentially labeled with distinct fluoro
phores, allowing them to be subjected to el
ectrophoresis as a single mixture and disti
nguished later using fluorometry.
Two methods to create nested set
s of DNA molecules

 DNA molecules are radioactively labe

led at their 5’ termini and are the
n subjected to four different regime
ns of chemical treatment that cause
them to break preferentially at Gs,
Cs, Ts, As. (no longer widely used)
 chain-termination (prevalent)
chain-terminating nucleotides

 The chain termination

method employ special
, modified substrates
called 2’-,3’-dideoxynu
cleotides (ddNTPs), w
hich once incorporate
d at the 3’ end of a gr
owing polynucleotide c
hain causes elongation
to terminate.
The chain termination method
The chain termination method

 We can read the full

nucleotide sequence
of the DNA by resolv
ing the four nested s
ets of fragments on
a polyacrylamide gel
.
Technical advancement

The chain termination method had und

ergone a series of technical adaptions
and improvements that allow the analy
sis of whole genomes.
Technical advancement

 Sequenator--- automated sequencing machin

e
 fluorescent chain-terminating nucleotide
s--- label each of the nested DNAs with a s
ingle “color”
9. Shotgun Sequencing a Bacteria
l Genome
 “shotgun” sequencing
1.The genome was randomly sheared into many
fragments with an average size of 1kb.
2.The pieces were cloned into plasmid recom
binant DNA vector.
3.DNA was prepared from individual recombin
ant DNA colonies and separately sequenced
on Sequenators.
“shotgun” sequencing

 In the method of “shotgun” sequenc

ing, every nucleotide in the genome
was sequenced ten times, which is kn
own as 10× sequence coverage.
 This method is more time consuming,
but is faster and less expensive.
Strategy for construction and sequenci
ng of whole genome libraries
The shotgun strategy permits a partial asse
mbly of large genome sequence

 HGP
ⅰ.DNA was prepared from each of the 23 chromosomes that co
nstitute the human genome, and then reduced into pools or
libriaries of small fragments using small-gauge pressuriz
ed needles. (typically, two or three libraries are constr
ucted for fragments of differing sizes)
ⅱ.These fragments were randomly cloned into bacterial plasm
ids
ⅲ.Recombinant DNA was isolated from bacterial plasmids and
then quickly sequenced using Sequenator (with an average
of 600 bp of DNA sequence per fragment, an average of two
million random DNA fragments are processed, that is one b
illion bp of sequence data)
ⅳ.Sophisticated computer programs assemble the shotgun sequ
ences into large contiguous sequences called contigs
The paired-end strategy permits the as
sembly of large genome scaffolds

ⅴ.Relatively short contigs are assembled i

nto larger scaffolds using paired-end seq
uencing
10.Genome-wide Analyses

 Finding protein coding genes in bact

eria and simple eukaryotes is relati
vely straightforward, essentially am
ounting to the identification of ORF
s.
 For animal genomes with complex exo
n-intron structures, the challenge i
s far greater.
Genome-wide Analyses
 A variety of bioinformatics tools are req
uired to identify genes and determine the
genetic composition of complex genomes.
 A notable limitation of current gene find
er programs is the failure to identify pr
omoters (such as TATA, INR, and DPE which
are noncoding exons)
 Computer programs should exploit more pro
perties of a gene: core promoter elements
, ORFs, splice sites etc. to identify pro
tein coding genes in a consistent and eff
ienct manner
11.Comparative Genome Analysis

 The comparisons of different animal

genomes not only permit a direct ass
essment of changes in gene structure
and sequence that arisen during evol
ution but refine the identification
of protein-coding genes within a giv
en genome.
Comparative Genome Analysis

 There is a high de
gree of synteny, c
onservation in gen
etic linkage, betw
een distantly rela
ted animals.
Comparative Genome Analysis

 Protein-coding seq
uences and regulat
ory sequences are
both tend to be co
nserved. But the i
dentification of r
egulatory sequence
s poses a greater
challenge.
Comparative Genome Analysis

 BLAST (basic local alignment search tool)

is a genome tool used to identify
 BLAST search shares a common feature of f
inding regions of similarity between diff
erent protein coding genes.
 A BLAST search can be done in several way
s
 One involves searching the genome or many
genomes for all of the predicted protein
sequences that are related to query seque
nce
Comparative Genome Analysis

Example of a
BLAST search
PROTEINS
1.Specific proteins can be purified
from cell extracts
 The purification of individual proteins is critical
to understanding their function.

The purification of a
protein is designed to
exploit its unique
characteristics,
including size, charge,
shape, and function.
2.Purification of a protein require
s a specific assay

 Incorporation assay (get DNA, RNA or

proteins labeled)are useful for monitoring
the purification and function of many diffe
rent enzymes catalyzing the synthesis of
polymers like DNA, RNA, or proteins.
3. Preparation of cell extracts con
taining active proteins

 Cell extracts can be lysed by detergent,

shearing forces, treatment with low ionic
salt or rapid changes in pressure.
 The goal is to weaken and break the me
mbrane surrounding the cell to allow prot
eins to escape.
4.Proteins can be separated from one ano
ther using column chromatography

 The two commonly used

methods – ion exchange
and gel filtration chromat
ography separate protein
s on the basis of their char
ge and size respectively.
5.Affinity chromatography can facilitate m
ore rapid protein purification

 Other reagents can be attached to colum

ns to allow the rapid purification of protei
ns, which is called affinity chromatogra
phy.
Immunoaffinity chromatography

 In this approach, an antibody that is spec

ific for target protein is attached to beads
. Ideally, this antibody will interact only wi
th the intended target protein. The bound
protein can then be eluted from the colu
mn using salt or mild detergent.
Immunoaffinity chromatography
 Proteins can be modified to facilitate thei
r purification, adding short additional ami
no acid sequences to the N-terminus or
C-terminus of a target protein.
 This modification can be generated using
molecular cloning methods or specific ep
itopes, which can be attached to any pro
tein.
6.Separation of proteins on polyacr
ylamide gels
 Sodium dodecyl sulphate (SDS)
 Electrophoresis in the presence of S
DS can be used to resolve mixtures o
f proteins according to the length o
f individual polypeptide chains.
 After electrophoresis, the proteins
can be visualized with a stain, such
as Coomassie brilliant blue
SDS-Polyacrylamide Gel Electrophores
is
7.Antibodies visualize electrophoretical
ly-separated proteins
 Immunoblotting
1. Electrophoretically separated proteins a
re transferred and bound to a filter
2. The filter is then incubated in a soluti
on of an antibody
3. The antibody finds the corresponding pro
tein on the filter to which it avidly bi
nds
4. A chromogenic enzyme is used to visualiz
e the filter-bound antibody.
Thank you