Genome Sequencing

Dr. P. Balaji
Vysya College, Hosur
 Introduction
 At the start of the 21st century the emphasis in molecular biology shifted from the
study of individual genes to the study of entire genomes.
 This chance in emphasis as prompted by the development during the 1990s of
methods for sequencing large genomes.
 Genome sequencing predates the 1990s that of the phage ф X174, was completed
on 1975 – but it was not until 20 years later, in 1995, that the first genome of a
free-living organism, the bacterium Haemophilus influenzae, was completely
sequenced.
 The next 5 years were a watershed with the genome sequences of almost 50 other
bacteria published, along with complete sequences for the much larger genomes of
yeast, fruit fly and humans.
 By 2000 the sequencing f bacterial genomes has become routine and projects
aimed at eukaryotic genomes were being approached with much greater confidence
than has been possible just a few years earlier. 
 Sequencing a genome
• A single chain termination sequencing experiment performed by hand produces
about 400 nucleotides of sequence, and a single run in an automated sequencer
gives about 750bp.

• But the total size of a fairly typical bacterial genome is 4000000 bp and the human
genomes is 3 200 000 000 bp.

• Clearly a large number of sequencing experiments must be carried out in order to

determine the sequence of an entire genome.

• The situation is not hopeless but it requires the use of robotic systems to prepare
the DNA for sequencing and t carry out the chain termination experiments, with the
sequences read by automated sequences that transfer the data directly into a
computer.

• In the factory-style laboratories that undertake these projects the main objective is
to keep the automated sequencers operating at their full capacities.

• Each sequencer can run up to 96 experiments in parallel, generation 72 000 bp of

sequence every two hours.
 Sequencing a genome

 The largest sequencing initiatives use up to 100 automated sequencers all working
around the clock, representing a theoretical output of 50 000 000 bp per day.

 Looked at in these terms, genome sequences are not so daunting after all. In
practice, the generation of sufficient sequence data is one of the more routine
aspects of a genome project.

 The first real problem that arises is the need to assemble the thousands or perhaps
millions of individual 750 bp sequences into a contiguous genome sequence.
 Strategies for sequence assembly

Two different strategies have been developed for sequence assembly.

 
The shotgun approach, in which the genome is randomly broken into short
fragments. The resulting sequences are examined for overlaps and these are
used to build up the contiguous genome sequence.
 
The clone contig approach, which involves a pre-sequencing phase during
which a series of overlapping clones is identified. Each piece of cloned
DNA is then sequenced, and this sequence placed at its appropriate position
on the contig map in order to gradually build up the overlapping genome
sequence.
 Shotgun approach
• The key requirement of the shotgun approach is that it must
be possible to identify overlaps between all the individual
sequences that are generated and this identification process
must be accurate and unambiguous so that the correct
genome sequence is obtained.
• An error in identifying a pair of overlapping sequences could
lead to the genome sequence becoming scrambled, or parts
being missed out entirely.
• The probability of making mistakes increases with larger
genome sizes, so the shotgun approach has been used mainly
wit the smaller bacterial genomes.
 The clone contig approach
 The clone contig approach does no suffer from the limitations of shotgun
sequencing and so can provide an accurate sequence of a large genome that contain
repetitive DNA.
 Its drawback is that it involves much more work and so takes longer and costs more
money.
 The additional time and effort is needed to construct the overlapping series of
cloned DNA fragments.
 Once this had been done, each cloned fragment is sequenced by the shotgun
method and the genome sequence built up step by step.
 The cloned fragments should be as long as possible in order to minimize the total
number needed to cover the entire genome.
 A high capacity vector is therefore used.
 The clone contig approach

 The first eukaryotic chromosome to be sequenced – chromosome III of

Saccharomyes cerevisiae – was initially cloned in a cosmid vector with the
resulting contig comprising 29 cloned fragments.
 Chromosome III is relatively short, however, and the average size of the cloned
fragments was just 10.8kb.
 Sequencing of the much longer human genome required 300 000 bacterial
artificial chromosome (BAC) clones.
 Assembling all of these into chromosome-specific contigs was a massive task.
 Gene Mapping

• Gene mapping is the assignment of a gene to a locus on chromosome.

• Mapping relative positions of many genes and other markers result into a
chromosome map.
• Essentially there are three types of gene map:
o Genetic (linkage) map
o Cytogenetic maps
o Physical (molecular) maps
Genetic map: Genetic or linkage map assigns genes to linkage groups (groups of
genes which tend to be inherited together because they are close together on
chromosome).

Cytogenetic map: correlating phenotypes with observable chromosome

rearrangemnts and deficiencies creates Cytogenetic map. This type of map has
allowed resolution and is applicable to few species like mammals and Drosophila,
which display either natural or artificially inducible reproducible chromosome
banding patterns. Cytogenetic maps if simple genomes (like viruses, plasmid) are
used to locate regions, which differ in their physical properties.

Physical map: Physical or molecular map is created by ordering cloned fragments of

genomic DNA. Physical map has the highest resolution and is the ultimate aim of
mapping projects.
 The purpose of gene mapping
 Gene mapping helps us to understand and exploit, the
inheritance of biological traits.

 Genetic linkage helps us to associate the inheritance of one

trait with another.

 It helps to correlate differences in phenotype with differences

in chromosome structure.

 Commercially, this helps plant and animal breeders to

produce crops or herds of improved qualities.

 In case of Human being, it helps assigning genes for genetic

diseases and their tracking through pedigrees by linkage to a
marker (gene tracking).
Polymerase Chain Reaction
The requirement for an oligonucleotide primer, and the constraint that
chain elongation must always occur in the 5' → 3' direction, make it
possible to obtain large quantities of a particular DNA sequence by
selective amplification in vitro.

The method for selective amplification is called the Polymerase Chain

Reaction (PCR).
Californian Kary B. Mullis was awarded a Nobel Prize in 1993
for his invention for PCR.

PCR amplification uses DNA polymerase and a pair of short,

synthetic oligonucleotide primers, usually 18–22 nucleotides in
length, that are complementary in sequence to the ends of the
DNA sequence to be amplified.
Role of primer sequences in PCR
amplification.
(A)Target DNA duplex (blue),
showing sequences chosen as the
primer-binding sites flanking the
region to be amplified.

(B)Primer (green) bound to

denatured strands of target
DNA.

(C)First round of amplification.

Newly synthesized DNA is shown
in pink. Note that each primer is
extended beyond the other
primer site.

(D)Second round of amplification

(only one strand shown); in this
round, the newly synthesized
strand terminates at the opposite
primer site.

(E)Third round of amplification

(only one strand shown); in this
round, both strands are
truncated at the primer sites.
Primer sequences are normally
about twice as long as shown
here.
Polymerase chain reaction
(PCR) for amplification of
particular DNA sequences.
Only the region to be amplified
is shown. Oligonucleotide
primers (green) that are
complementary to the ends of
the target sequence (blue) are
used in repeated rounds of
denaturation, annealing, and
DNA replication. Newly
replicated DNA is shown in
pink. The number of copies of
the target sequence doubles in
each round of replication,
eventually overwhelming any
other sequences that may be
present.
 Reaction components
1. Taq polymerase
2. Oligonucleotide primers – two
3. Buffers – Tris buffer of pH 8.8 at 25˚ C gives pH 7.3 at 72˚ C.
4. Mg++ required by Taq enzyme activity.
5. Detergents (non-ionic - (triton X – 100) is added both in storage solution
and in reaction mixture. Taq is highly hydrophobic protein and tends to
precipitate form aqueous solution. Detergents added help maintain their
activity.
6. Salts other than Mg++ are not at all required. KCl, phosphates are
inhibitory.
7. Nucleotides – 800μM of total dNTPs added in reaction mixture. This is
sufficient to produce 1.3μg of DNA in 150μl reaction.
8. 2 mercaptoenthanol is added to PCR mixture to stabilize protein during
thermal cycling. Carrier proteins (or) agarose is added but is not necessary
and may be interfering at times.
9. Template DNA – Sample DNA need not be pure. Even less than 2μg is
sufficient.
 Enzyme used in polymerase chain
reaction
 Implementation of PCR with conventional DNA polymerases is not
practical, because at the high temperature necessary for denaturation,
the polymerase is itself irreversibly unfolded (denatured) and becomes
inactive.

 However, DNA polymerase isolated from certain organisms is heat

stable because the organisms normally live in hot springs at
temperatures well above 90°C, such as are found in Yellowstone
National Park.

 Such organisms are said to be thermophiles.

 The most widely used heat-stable DNA polymerase is called Taq

polymerase, because it was originally isolated from the thermophilic
bacterium Thermus aquaticus.
M  The primers are oriented with their 3' ends pointing in the direction of the
region to be amplified, because each DNA strand elongates only at the 3' end.
After the primers have annealed, each is elongated by DNA polymerase using

et
the original strand as a template, and the newly synthesized DNA strands (red)
grow toward each other as synthesis proceeds.

 To start a second cycle of PCR amplification, the temperature is raised again to

h denature the duplex DNA. Upon lowering of the temperature, the original
parental strands anneal with the primers and are replicated.

o
 The daughter strands produced in the first round of amplification also anneal
with primers and are replicated.

 In this case, although the daughter duplex molecules are identical in sequence

d to the original parental molecule, they consist entirely of primer

oligonucleotides and nonparental DNA that was synthesized in either the first
or the second cycle of PCR.

s  As successive cycles of denaturation, primer annealing, and elongation occur,

the original parental strands are diluted out by the proliferation of new
daughter strands until eventually, virtually every molecule produced in the
PCR
M
et • The power of PCR amplification is that the number of copies of the
template strand increases in exponential progression: 1, 2, 4, 8, 16, 32,

h 64, 128, 256, 512, 1024, and so forth, doubling with each cycle of
replication.

o • Starting with a mixture containing as little as one molecule of the

fragment of interest, repeated rounds of DNA replication increase the

d number of amplified molecules exponentially.

s
M • For example, starting with a single molecule, 25 rounds of DNA
replication will result in 225 = 3.4 X 107 molecules.

et • This number of molecules of the amplified fragment is so much greater

than that of the other unamplified molecules in the original mixture that
the amplified DNA can often be used without further purification.

h • For example, a single fragment of 3 kb in E. coli accounts for only 0.06

percent of the DNA in this organism.

o • However, if this single fragment were replicated through 25 rounds of

replication, then 99.995 percent of the resulting mixture would consist

d
of the amplified sequence.

• A 3-kb fragment of human DNA constitutes only 0.0001 percent of the

total genome size.

s • Amplification of a 3-kb fragment of human DNA to 99.995 percent

purity would require about 34 cycles of PCR.
Why “Polymerase”?

It is called “polymerase” because the

only enzyme used in this reaction is
DNA polymerase.
Why “Chain”?

It is called “chain” because the

products of the first reaction become
substrates of the following one, and
so on.
The “Reaction” Components

1) Target DNA - contains the sequence to be amplified.

2) Pair of Primers - oligonucleotides that define the sequence to be

amplified.

3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.

4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction

5) Mg++ ions - cofactor of the enzyme

6) Buffer solution – maintains pH and ionic strength of the reaction

solution suitable for the activity of the enzyme
The Reaction

PCR tube THERMOCYCLER

 PCR optimization
 PCR reactions are not usually 100% efficient, even when using cloned
DNA and primers of defined sequence.

 Usually the reaction conditions must be varied to improve the

efficiency.

 This is very important when trying to amplify a particular target from a

population of other sequences, for example one gene from genomic
DNA, or one cDNA from either a cDNA library or the products of a
first strand cDNA synthesis reaction.

 This latter method of reverse transcribing mRNA and then PCR

amplifying the first strand cDNA is called reverse transcriptase (RT)-
PCR.

 If the reaction is not optimal, PCR often generates a smear of products

on a gel rather than a defined band.
 PCR optimization
• The usual parameters to vary include the annealing temperature and
the Mg2+ concentration.

• Too low an annealing temperature favors mispairing.

• The optimal Mg2+ concentration varies with each new sequence, but is
usually between 1 and 4 mM.

• The specificity of the reaction can be improved by carrying out nested

PCR, where, in a second round of PCR, a new set of primers are used
that anneal within the fragment amplified by the first pair, giving a
shorter PCR product.
 
• If on the first round of PCR some nonspecific products have been
produced, giving a smear or a number of bands, using nested PCR
should ensure that only the desired product is amplified from this
mixture as it should be the only sequence present containing both sets
of primer-binding sites.
 An overview of the polymerase chain
reaction
• In the first cycle of PCR amplification, the DNA is denatured to
separate the strands. The denaturation temperature is usually around
95°C.

• Then the temperature is decreased to allow annealing in the presence of

a vast excess of the primer oligonucleotides.

• The annealing temperature is typically in the range of 50°C–60°C,

depending largely on the G + C content of the oligonucleotide primers.

• To complete the cycle, the temperature is raised slightly, to about 70°C,

for the elongation of each primer.

• The steps of denaturation, renaturation, and replication are repeated

from 20–30 times, and in each cycle the number of molecules of the
amplified sequence is doubled.
 Denature (heat to
95oC)

Lower temperature to 56oC

Anneal with primers

Increase temperature to
72oC DNA polymerase +
dNTPs
 DNA copies vs Cycle number
2500000

2000000
DNA copies

1500000

1000000

500000

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Cycle number
 Problems and
 Contamination of the reaction mixture of PCR Limitations
even by a single gene
can result into impure product of PCR reaction.

 If diagnosis of male specific genes is to be done, female workers are

required otherwise cells of skinof male workers can also cause
contamination.

 PCR cannot be performed directly on food samples and processing of

sample is required to extract DNA to be studied.

 PCR is most useful for the amplification of DNA segment less than 2kb
in length.

 Base substitution error by Taq polymerase is 1 / 9,000 base pair and

frame shifts occurs 1 / 40,000.

 There are significant if PCR extends over 30 cycles when error occurs
in every 300basepair of product. High error rate of Taq is because it
lacks 3’ -5’ exo nuclease activity (proof reading).
 Application

 Classification of organisms
 Genotyping
 Molecular archaeology
 Mutagenesis
 Mutation detection
 Sequencing
 Cancer research
 Detection of pathogens
 DNA fingerprinting
 Drug discovery
 Genetic matching
 Genetic engineering
 Application
o PCR amplification is very useful for generating large quantities of a specific
DNA sequence.

o The principal limitation of the technique is that the DNA sequences at the ends of
the region to be amplified must be known so that primer oligonucleotides can be
synthesized.

o In addition, sequences longer than about 5000 base pairs cannot be replicated
efficiently by conventional PCR procedures, although there are modifications of
PCR that allow longer fragments to be amplified.

o On the other hand, many applications require amplification of relatively small

fragments.

o The major advantage of PCR amplification is that it requires only trace amounts
of template DNA.
 Application
o Theoretically only one template molecule is required, but in practice the
amplification of a single molecule may fail because the molecule may, by
chance, be broken or damaged.

o But amplification is usually reliable with as few as 10–100 template molecules,

which makes PCR amplification 10,000–100,000 times more sensitive than
detection via nucleic acid hybridization.

o The exquisite sensitivity of PCR amplification has led to its use in DNA typing
for criminal cases in which a minuscule amount of biological material has been
left behind by the perpetrator.

o PCR is widely used in the study of independent mutations in a gene whose

sequence is known in order to identify the molecular basis of each mutation, to
study DNA sequence variation among alternative forms of a gene that may be
present in natural populations, or to examine differences among genes with the
same function in different species.
 Application
o The PCR procedure has also come into widespread use in
clinical laboratories for diagnosis.

o To take just one very important example, the presence of

the human immunodeficiency virus (HIV), which causes
acquired immune deficiency syndrome (AIDS), can be
detected in trace quantities in blood banks via PCR by
using primers complementary to sequences in the viral
genetic material.

o These and other applications of PCR are facilitated by the

fact that the procedure lends itself to automation by the use
of mechanical robots to set up and run the reactions.
 Application

Some of the key applications of PCR include:

 
1.Diagnosis of diseases by detection of bacteria / virus.
2.Diagnosis of genetic diseases.
3.DNA finger printing for forensic medicine.
4.For research purpose.
5.For molecular mapping.
6.For preparation of molecular marker.
7.To monitor genetic engineering and gene therapy experiments
 Summar
y

blood, chorionic
villus, amniotic 68,719,476,736 copies Gel Analysis,
fluid, semen, hair Restriction
root, saliva Digestion,
Sequencing
Conclusion
The speed and ease of use,
sensitivity,
sensitivity specificity and
robustness of PCR has
revolutionised molecular biology
and made PCR the most widely
used and powerful technique with
great spectrum of research and
diagnostic applications.
Biology of Genetic Engineering
Once recombinant DNA molecules have been constructed in vitro, the
desired sequence can be isolated.

Three things have to be done to achieve this:

(i)The individual recombinant molecules have to be physically separated

from each other

(ii)The recombinant sequences have to be amplified to provide enough

material for further analysis and

(iii)The specific fragment of interest has to be selected by some sort of

sequence-dependent method.

The biology of gene cloning, which involves the use of a

suitable carrier molecule or vector, and requires a suitable
living system or host in which the vector can be
propagated.
 Host cell types

• The type of host cell used for a particular application will depend
mainly on the purpose of the cloning experiment.
• If the aim to isolate a gene for structural analysis, the requirements
may calla for a simple system that is easy to use.
• If a aim is to express the genetic information in a higher eukaryote
such as plant, a more specific system will be required.
• These two aims are not necessarily mutually exclusive; often a simple
primary host is used to isolated a sequence that is then introduced into
a more complex system for expression.
 Prokaryotic hosts – E.
 coli
An ideal host cell should be easy to handle and propagate, should be
available as a wide variety of genetically defined strains, and should
accept a range of vector.

 The bacterium E. coli fulfils these requirements. And is used in many

cloning protocols.

 E. coli has been studied in great detail and many different strains were
isolated by microbial geneticists as they investigated the genetic
mechanisms of this prokaryotic organisms,.

 Such studies provided the essential background information on which

genetic engineering is based.

 E. coli is a G-ve bacterium with a single chromosome packed into a

compact structure known as the nucleoid.

 The genome size is some 4 x 106 base pairs.

 Prokaryotic hosts – E.
• There are, however, certain drawbacks with most of these.
coli
• Often there are few suitable vectors available for use in such cells and
getting recombinant DNA into the cell can cause problems.

• This is particularly troublesome when primary cloning experiments are

envisaged, i.e., direct introduction of ligated recombinant DNA into the
host cell.

• It is often more sensible to perform the initial cloning in E. coli, isolate

the required sequence, and then introduce the purified DNA into the
target host.

• May of the drawbacks can be overcome by using this approach,

particularly when vectors that can function in the target host and in E.
coli (shuttle vectors) are used.
 Eukaryotic hosts – Yeast
 One disadvantage of using an organism such as E. coli as a host for
cloning is that it is a prokaryote, and therefore lacks the membrane-
bound nucleus (and other organelles) found in eukaryotic cells.
 This means that certain eukaryotic gees may not function in E. coli as
they would in their normal environment, which can hamper their
isolation by selection mechanisms that depend on gene expression.
 Also, if the production a eukaryotic protein is the desired outcome of a
cloning experiment, it may be base easy to ensure that a prokaryotic
host produces a fully functional protein.
 Eukaryotic ells range from microbes, such as yeast ad algae, to cells
from complex multi-cellular organisms, such as ourselves.
 The microbial cells have many of the characteristics of bacteria with
regard to ease of growth and availability of mutants.
 Eukaryotic hosts – Yeast
 Higher eukaryote present a different set of problems to the genetic
engineer, may of which require specialized solution.
 Often the aim of a cloning experiment in a higher plant or animal is to
alter the genetic makeup of the organism,. Rather than to isolate a gene
for further analysis or to produce large amounts of a particular protein.
 The yeast Saccharomyces cerevisiae is the favored eukaryotic microbe
for genetic engineering.
 It has been used for centuries in the production of bread and beer and has
been studied extensively.
 The organism is amenable to classical genetic analysis and a range of
mutant cell types is available.
 In terms of genome complexity, Saccharomyces cerevisiae has about
1.35 x 107 base pairs of DNA, some 3.5 time more than E. coli.
 Eukaryotic hosts – Yeast

 Other fungi that may be used for gene cling experiments include
Aspergillus indulans and Neurospora crassa.
 Plant and animal cells may also be used as hosts for gene manipulation
experiments.
 Unicellular algae such as Chlamydomonas reinhardtii have all the
advantages of microorganisms plays the structural and functional
organization of plant cells, and their use in genetic manipulation will
increase as they become more widely studied.
 Other plant cells (and animal cells) are usually grown as cell cultures,
which are much easier to manipulate than cells in a whole organism.
Plasmids
1. Plasmids are small, circular, double stranded, extra chromosomal DNA’s
present in bacterial cells.
2. They are inherited sharply without the influence of chromosomal DNA.
3. They replicate independently due to the presence of an “Origin of
Replication”.
4. The Plasmids are 1 Kbp – 200 Kbp in size and have limited number of
genes.
5. Most bacteria contain more than one copy of each plasmid (Copy number).
6. The copy number of plasmids usually varies from 1-50.
7. The copy number can be increased by treating bacterial culture with
chloramphenicol
A number of host properties are specified by plasmids, such
as
Antibiotic and Heavy metal resistance
Nitrogen fixation
Pollutant degradation
Bacteriocin production
Toxin production
Colicin factors
The plasmids typically have three important elements:

A cloning site (a place to insert foreign DNAs),

An origin of replication.

A selectable marker gene (e.g. resistance to ampicillin)

Plasmids may also be classified on the basis of the number of
copies found in the host cell.
Low copy number (1-4 copies per cell).

Moderate copy number (10 – 100 copies per cell).

High copy number (>100 copies per cell).

 The copy number of plasmid is generally defined as the ratio of number of
moles of plasmid DNA to the number of mole equivalents of chromosomal
DNA.
 Copy number is a parameter of a bacterial population and may vary around
mean value.
 Low copy number plasmids exhibit stringent control of DNA replication with
replication of pDNA closely tied to host cell chromosomal DNA replication.
 Conjugative plasmids are large with stringent control of DNA replication and
are present in low copy number.
 Non-conjugative plasmids are small, high copy number and have relaxed
control of DNA replication.
 High copy number plasmids are useful for expression of cloned gene to get
higher yields.
Plasmids are also designated as
 Plasmid
1.
incompatibility
Some microorganisms may have as many as 8-10 different plasmids.
2. Plasmid incompatibility is the inability of two different plasmids to
coexist in the same host cell in absence of selection pressure.
3. Groups of plasmids, which are mutually incompatible, are considered to
belong to the same incompatibility group.
4. Currently 30 incompatibility gropes are defined for E. coli plasmids and
13 for Staphylococcus aureus-plasmids.
5. Plasmids belonging to incompatibility classes P, Q and W are termed as
promiscuous and they can enter into wide host range and can be
maintained stably in them.
6. The promiscuous plasmids can potentially transfer cloned DNA in
between diverse hosts.
 Importance as cloning vectors
 Plasmids have basic attributes like origin of replication and
self transfer capacity and selectable markers to act as
potential vectors.
 But plasmids also lack some important features required
for high quality cloning vector.
 The drawbacks are: (a) small size and efficiency to transfer
maximum of 15 kb of foreign DNA. (b) Single restriction
endonucleases site, (c) one or more selectable genetic
markers.
 But these drawbacks have been overcome by genetically
engineering the natural plasmid into construction of
plasmid cloning vectors.
 Copy number of the plasmids, which are used as vector,
can be increased by amplification.
 Importance as cloning vectors
 In presence of inhibitors of protein synthesis, such as
chlormaphenicol or spectromycin, chromosomal
replication is inhibited whereas replication of the
plasmid continues.
 Runaway-replication plasmids are vectors, which have
conditionally controlled replicons.
 For example a mutant of plasmid R1 at non-permissive
temperatures (300C) have 20-30 copies while above
350C, they are 75% of total DNA of the cell.
 The useful expression period can be prolonged by
limiting this amplification by addition of sub-lethal
concentrations of inhibitors like novobiocin.
 Natural plasmids are those plasmids, which are not
constructed in vitro for the sole purpose of cloning.
 Importance as cloning vectors
 Examples of natural plasmids, which are used for cloning
DNA, are pSC 101, col E1 and RSF 2124.
 Suicide plasmids are the plasmid derivatives which are
used for introducing the transposable element into cells
but he plasmids are incapable of replicating in the
recipient (replication of plasmid is locked due to
mutation affecting replication.
 Carriage of plasmids imposes a burden upon the host.
 Plasmid free cells arise during the growth and ultimately
overgrow plasmid-containing cells.
Types of plasmids:
 
On the basis of conjugative transfer, Plasmids are classified
into 2 categories.
 
They are –
A) Conjugative Plasmids. These plasmids are transferred
from one bacterium to the other during conjugation. They
contain “ tra genes” for the conjugative transfer. Eg F-
Plasmid.
B) Non conjugative Plasmids. These plasmids do not pass
from one bacterium to another bacterium during conjugation.
E.g. col E1 Plasmid.
On the basis of the functions, plasmids are classified into five
types. They are
A) F Plasmid: F Plasmid or fertility plasmid contains some
genes expressing the maleness in bacteria. The genes are
known as “tra genes”.
B) R plasmid: R Plasmid (Resistance plasmids) contain some
genes giving resistance to bacteria against antibiotics and heavy
metals. E.g pSC 101 gives organism tetracycline resistance.
C) Col plasmid: Col Plasmids code for the synthesis of
bacterial toxin Colicin. Colicin kills other closely related strains
of bacteria. E.g Col E1, Col B, etc.
D) Degradative Plasmids: Degradative plasmids code for
enzymes that degrade toxic substances such as toluene, xylene,
salicylic acid, parathion etc. Tol Plasmid of Pseudomonas putida
involves in the breakdown of toluene.
E) Virulence Plasmids: Virulence plasmids provide
pathogenicity to bacteria.
On the basis of Copy number:
A) Stringent Plasmid: Which have limited number of copies per
cell.(1-2 Nos).
B) Relaxed Plasmid: Which are normally maintained at multiple
copies per cell (20 – 50)
 
 Based on the Occurrence
 A) Natural Plasmid: Some plasmids are isolated from bacteria
and directly used for gene cloning without any modification. Such
plasmids are known as Natural plasmid vectors. Eg RP4 plasmid of
Pseudomonas, col E1 of E.coli and Yep and YIp of yeasts.
 
B) Natural based plasmid vectors: The effective cloning vectors
created from wild type plasmids are called Based plasmid vectors,
Constructed plasmid vectors, derived vectors or artificial vectors.
Eg pBR 322, RSF 1010, pSC 101 and pUC 8 etc.
Plasmid as cloning vectors:
 
A.Chang and N.Cohen (1973) first proved the use of
plasmids as gene cloning vectors.
They isolated plasmids from two different strains of
bacteria and fused them using restriction enzyme and DNA
ligase.
The fused DNA (chimeric plasmid) was then introduced into
E.coli cells and its expression was studied.
They found out that the genes of both the plasmids express
their traits in E.coli.
Properties of plasmid
The ideal cloning vectors must have the following characteristic features.
1. Size
The plasmid must be small in size.
The small size is helpful for easy uptake of chimeric DNA by host cells and
for the isolation of plasmid without damage.
2. Copy number
The plasmid must be present in multiple copies.
3. Genetic markers
The plasmid must have one or a few genetic markers. These markers
help us for the selection of organism that has recombinant DNA.
4. Origin of replication
The plasmid must have its own origin of replication and regulatory genes
for self – replication.
5. Unique restriction sites
The plasmid must have unique restriction sites for RE in marker genes.
6. Insertional inactivation
This will help us for the selection of recombinants by insertional
inactivation method.
7. Pathogenicity
The plasmid should not have any pathogenic property.
Distribution of plasmids among Bacterial species
 Plasmids are present among diverse group of bacteria.
1.They are found in
1. Phototrophic bacteria
2. Cyanobacteria
3. Gram-positive, Gram-negative, filamentous
endospore forming and in extreme thermophilic
bacteria.
Plasmid classification 
Classification by structure

Classification by phenotype

Classification on the basis of intrinsic properties.

Classification by structure:
Single stranded circular DNA plasmid. Eg Streptomyces
and Clostridium species.
Linear double stranded DNA plasmids.
Ds closed circle plasmids
RNA plasmids – Viriods are specialized in Ss circular RNA
plasmids.
Bipartite, linear, Ds RNA elements found in yeast. They
are called ‘Killer factors’ as they confer killer phenotype
upon the host.
Classification by phenotype
In bacterial plasmids, genes which are non essential for

replication, maintenance or transfer are often carried on

mobile elements which can transpose to other plasmids and

unto host chromosome.

Thus phenotype conferred by a bacterial plasmid does not

reflect any intrinsic property of plasmid.

Classification on the basis of intrinsic properties:
A better plasmid classification system uses intrinsic
properties such as transfer, replication and maintenance
mechanism.
Plasmid transfer occurs by 4 routes.
1. Cell fusion
2. Transformation
3. Transduction
4. Conjugation
First 3 route are passive transfer. Last one is active transfer
Suicide plasmid
The plasmid derivatives which are used for introducing

the transposable element into cells but the plasmids

are incapable of replicating in the recipient.

Plasmid forms
 
Plasmids are double stranded circular DNA’s. They can exist
in following different forms
1. Covalently closed circles (CCC) – both strands of DNA
intact.
2. Open circles (OC) – Only one of the 2 strands is
intact.
3. Linear
4. Super coiled.