Terry Brown
Genomes
Third Edition
Chapter 2:
Studying DNA
Copyright © Garland Science 2007
Chapter Objectives
• Understand PCR and its application in
genomics as well as PCR based cloning.
• Clearly understanding of various enzymes
and their function for genomic research.
• Key features of cloning vectors.
• Relationship of vector-host system and their
application in genome library construction.
Tool Kit to study Genomics
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DNA is invisible to naked eye
To manipulate DNA as desired way we
need certain tools which can be
effectively utilized to study genomes
The research during 1970, and 1980s
resulted in the development of assembly
of tools which can be utilized to work
with DNA
Researcher identified and purified
different enzymes which can:
– Cut DNA
– Ligate DNA
– Copy DNA
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This ability gave birth to recombinant DNA
technology where DNA from different sources can
be combined in a way that never existed before!
Recombinant DNA technology led to the
development of DNA/Gene cloning where a piece
or gene of interest can be manipulated in virtually
any host
– Making it possible to know the function of a
gene in a model host
Enzymes for DNA manipulation
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DNA polymerases
Nucleases
Ligases
End-Modification
enzymes
DNA polymerases
• Replicate the genome
• Template-dependent DNA
polymerase
• Need primers
• 3’-5’ Exonuclease activity (proof
reading)
• 5’-3’ Exonuclease activity (Primer
removal)
Figure 2.2 Genomes 3 (© Garland Science 2007)
DNA polymerases in Research
• First DNA pol I identified in E.coli by Kornberg and named as
Kornberg Polymerase (contains both exonuclease activities)
• A modified version of Kornberg Pol is called as Klenow Pol which
lacks 5’-3’ exonucleases activity used for sequencing initially.
• Thermostable DNA polymerases Taq Pol
• Sequenase for Sequencing
• Sequenase is used for sequencing due to
its properties like:
– High processivity
– Negligible or zero 5’-3’ activity
– Negligible or zero 3’5’ activtiy
• RNA dependent DNA polymerases:
– Synthesize cDNAs
Nucleases
• The nucleases are very important and has DNA or RNA cutting ability
• They fall into two classes
– Exonucleases
– Endonucleases
• restriction endonucleses
Restriction endonucleases Enable DNA molecules
to be cut at defined positions
• There are three types of restriction
endonucleses
– Type I and III: (bind at specific
sites but cut at different sites)
– Type II: bind and cuts at specific
sites
• Produces blunt or flush ends
• Produces sitcky or cohesive
ends
Figure 2.5 Genomes 3 (© Garland Science 2007)
Examining the results of a
restriction digest
• The results of Restriction digestions are analyzed by
– Agarose gel electrophoresis
– Southern hybridization
Southern hybridization
Figure 2.6b Genomes 3 (© Garland Science 2007)
DNA Ligases
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Two DNA fragments can be joined together by
the help of DNA ligases which join them by
introducing phophodiester bond.
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Most widely used DNA ligase is from E.coli
infected with T4 phage
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Utilize ATP
NAD
Involve in T4 replication
The efficiency of ligation depends on
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Concentration of DNA
The probability of two ends to come closer to each other
so that ligase can work
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The efficiency can be increased by the utilization
of:
– Linkers
• Are short fragment of dsDNA
containing a site for restriction
endonuclease
• Which will produce sticky ends after
its digestion to facilitate cloning
– Adoptors
• Short fragment of dsDNA which have
one end blunt and other end is already
sticky so no need of digestion
– Homopolymer tailing by terminal
deoxynucleotidyle
• Adds non template nucleotides on the
3’ end of DNA
• Any polynuclotides can be added like
poly G to facilitate cloning with poly C
End Modification Enzymes
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Terminal Deoxynucleotidyl Transferase
– Obtained from calf thymus tissue
– Used for homopolymer tailing
Alkaline phophatase
– Obtained from calf thymus tissue and E.coli
– Removes phophate groups from the 5’ end of DNA
– This prevents self ligation of the molecules
T4 polynucleotide kinase
– Obtained from T4 infected E.coli
– Performs reverse action of alkaline phophatase
– It adds phophates to 5’-ends
The judicious use of alkaline phophatase and T4 polynucleotide kinase
direct the action of DNA ligase in a predictive way
DNA Isolation
• Chemical purification
• Ion exchange purification of DNA
The Polymerase Chain Reaction
(PCR)
• PCR is a very powerful technique
• Its impact on our understanding of genes and genomes has
been immeasurable
• PCR complements DNA cloning
• Helps in purification of a segment of DNA by its
amplification
• The cloning works without knowing the sequence of DNA
• While PCR has limitation that for utilizing PCR one need
to know the flanking region of target DNA
Carrying out a PCR
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The enzyme: thermostable DNA polymerase from
Thermus aquaticus
Reaction mixture:
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Buffer to maintain pH, salt and composition, medium to
dissolve DNA and enzyme
Enzyme
Primers
Template
MgCl2
dNTPs
water
Conditions:
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Denatureing
Annealing
Extension
DNA Cloning
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A process to ligate the DNA in a specific
background
– To have identical copies
– To place the DNA in a new genetic
background
– To change the already present gene
The technique was developed due to the
ability to:
– Cut the DNA
– Ligate it in some other place
The cloning was first invented in early
1970s and has revolutionized the field of
molecular biology
Cloning Vectors
• The plasmids act as cloning vectors
• They contains some genetic characters
with provides some advantages like:
– Origin of Replication
– Selectable Marker
– Screening Marker
Vectors Based on E. Coli
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The simplest vectors
Can be introduced into E. coli by the process of transformation
The most popular plasmid vector is pUC18 introduced in 1980s.
– 2.7 kb
– Origin of replication
– Two important genes
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Selectable marker (ampicillin resistance gene) which produce
β-latamase enzyme which breakdown the ampicillin
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Secreening marker (LacZ’ segment of LacZ gene which
produce β-galactosidase).
– The lacZ’ produces α-peptide portion of β-galactosidase
enzyme
– This vector need to transformed in those E.coli strains which
have modified LacZ gene and only can produce βgalactosidase when LacZ’ is present on plasmid enabling
screening based on insertional inactivation
– Develop blue color in the presence of X-Gal (5-bromo-4chloro-3-indolyl-B-D-galactopyranoside).
• Limitations
– Can only accommodate small sized DNA
like 10 kb at max
– Larger segments interfere with plasmid
replication.
– Some sequences which are
complementary to each other making
secondary structure are not well handled
by some strains of E. coli
Cloning vectors based on E. Coli
bacteriophage genome
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Bacteriophage are viruses which infects E.
coli and replicate in them.
They are have two different type of mode of
infection cycles of bacteriophage λ
– Lytic infection cycle
• Infect cell, multiply and breakdown
the cell to release out
– Lysogenic infection cycle
• Infect cell, integrate in the bacterial
genomic DNA, multiplicate as E.
coli do, in some cell starts lytic cycle
after excision from host DNA
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The bacteriophage λ vectors can accommodate more DNA like 15 kb
The size of λ genome is 48.5 kb
– 15 kb segment contains genes needed for integration of phage DNA into host genome.
– This portion which is “optional” can be deleted from its genome without impairing
phage ability to replicate and synthesize new phage particles.
– This portion can be utilized for cloning foreign DNA fragments
– The linear genome of λ genome contains 12 nucleotides single stranded overhangs,
called cos sites, which have complementry sequences and can base pair each others
Insertional vectors
– In which optional region has been removed an a unique restriction site introduced at
some position for cloning
Replacement vectors
– In which optional regions remains within a stuffer fragment , which is flanked by a pair
of restriction sites for replacement with desired fragment.
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The cloning in λ genome can be done in two ways
– Recircularization of genome by the help of cos
sites and reintroduce it to E. coli by
transfection
– By in vitro packaging system which its cutting
into tow arms i.e. left arm and right arm
– Then the DNA is ligated with these arms
– The successful clone will contains left and
right arm at the outer side of cloned fragment
– Only those DNA can be packed which have
• Size of 37-52 kb
• Containing cos sites at both sides
The in vitro packaged viruses are used to infect E.
coli which ultimately forms plaque from where be
further analyzed for the presence of DNA
– Blue white screening system can also be used with
it.
Vectors for Longer Pieces of
DNA
• In λ phage particles 18 kb fragment of DNA can be cloned
• Its higher than plasmid based vector but still very small compared with
the sizes of intact genomes.
• Smaller the cloning size larger the number of clones that would be
required to make a genomic library
Cosmid Vectors
• Cosmid is a plasmid carrying λ cos site as the
end.
• Concatamers of cosmid molecules, linked at their
cos sites, act as substrates for in vitro packaging.
• So the particles containing cosmid DNA are
infective but can not make new phage particles
• But comsid can replicate due to the presence of
ori in side the cell as plasmid.
• About 44 kb of DNA fragment can be cloned into
cosmids
Yeast Artificial Chromosomes
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The development of YACs lead to the major breakthrough for
cloning DNA fragments having more than 50 kb size.
These vectors are propagated in yeast i.e. saccharomyces cerevisiae
YACs are based on chromosomes rather than plasmids or virueses.
YACs contains three important components
– The centromere (role in cell division)
– Telomeres (important for protection of the ends)
– Origins of replication (one or more for initiation of replication)
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All these functionally important regions make about 10-15 kb DNA, while
yeast chromosomes range in size from 230 to 1700 kb.
– There is a potential to clone Mb size DNA fragments in YAC
– The standard YACs 600 kb DNA
– But there are associated some problem of DNA instability
Other vectors
• Bacterial artificial chromosomes
– Based on F plasmid of e.coli
– Higher capacity of larger DNA
– Lac selection
– 300 kb can be cloned in them
– Very famous and been used in Human Genome Project
• Bacteriophagte P1 Vectors
– Very similar to lambda vectors, but P1 phage has larger genome
– Can clone 125 kb DNA
• P1-derived artificial chromosomes (PAC)
– Combine features of P1 and BACs
– Can clone 300 kb
• Fosmids
– Contains the F plasmid origin of replication and
cos sites
– Less prone to instability problems
Cloning in organisms other than
E. coli
• Cloning is not only useful for DNA coping rather it is used for
expression analysis
– For understanding the regulation
– For producing things in other organisms like insulin
• Vectors are developed for yeast and fungus
– Based on 2 um circle a yeast plasmid
– I.e. YIp5 vector (yeast integrative plasmid)
– Its shuttle vector can replicate in e.coli and yeast
– Contains a gene URA3 (uracil production pathway enzyme)
Ti Plasmid
• From soil microorganism Abgrobacterium
Tumefaciens.
– For plant transformation
– Ti plasmid contains T-DNA which integrates
into host genome
– Binary vector are now developed on it
– Integrates into host genome for stable
transformation