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UNIT I –TOOLS AND TECHNIQUES OF GENETIC ENGINEERING
ENZYMES
Restriction Endonucleases— DNA Cutting Enzymes:
Restriction endonucleases are one of the most important groups of enzymes for the
manipulation of DNA. These are the bacterial enzymes that can cut/split DNA (from any source)
at specific sites. They were first discovered restricting the replication of bacteriophages in
E.coli, by cutting the viral DNA (The host E.coli DNA is protected from cleavage by addition of
methyl groups). Thus, the enzymes that restrict the viral replication are known as restriction
enzymes or restriction endonucleases.
Hundreds of restriction endonucleases have been isolated from bacteria, and some of them are
commercially available. The progress and growth of biotechnology is unimaginable without the
availability of restriction enzymes.
Nomenclature:
Restriction endonucleases are named by a standard procedure, with particular reference to the
bacteria from which they are isolated. The first letter (in italics) of the enzymes indicates the
genus name, followed by the first two letters (also in italics) of the species, then comes the
strain of the organism and finally a Roman numeral indicating the order of discovery. A couple
of examples are given below.
EcoRI is from Escherichia (E) coli (co), strain Ry13 (R) and first endonuclease (I) to be discovered.
Hindlll is from Haemophilus (H) influenza (in), strain Rd (d) and the third endonucleases (III) to
be discovered.
Types of endonucleases:
At least 4 different types of restriction endonucleases are known-type 1 (e.g Ecok12), type II
(e.g. EcoRI), type III (e.g. EcoPI) and type IIs. Their characteristic features are given in Table 6.1.
Among these, type II restriction endonucleases are most commonly used in gene cloning.
Recognition sequences:
Recognition sequence is the site where the DNA is cut by a restriction endonuclease. Restriction
endonucleases can specifically recognize DNA with a particular sequence of 4-8 nucleotides and
cleave. Each recognition sequence has two fold rotational symmetry i.e. the same nucleotide
sequence occurs on both strands of DNA which run in opposite direction (Table 6.2). Such
sequences are referred to as palindromes, since they read similar in both directions (forwards
and backwards).
Cleavage patterns:
Majority of restriction endonucleases (particularly type II) cut DNA at defined sites within
recognition sequence. A selected list of enzymes, recognition sequences, and their products
formed is given in Table 6.2. The cut DNA fragments by restriction endonucleases may have
mostly sticky ends (cohesive ends) or blunt ends, as given in Table 6.2. DNA fragments with
sticky ends are particularly useful for recombinant DNA experiments. This is because the single-
stranded sticky DNA ends can easily pair with any other DNA fragment having complementary
sticky ends.
Reverse transcriptase
An enzyme, occurring in retroviruses, that catalyses the formation of double-stranded DNA
using the single RNA strand of the viral genome as template.
The enzyme is used in genetic engineering for producing complementary DNA from
messenger RNA.
DNA Ligases —DNA Joining Enzymes:
The cut DNA fragments are covalently joined together by DNA ligases. These enzymes were
originally isolated from viruses. They also occur in E.coli and eukaryotic cells. DNA ligases
actively participated in cellular DNA repair process.
The action of DNA ligases is absolutely required to permanently hold DNA pieces. This is so
since the hydrogen bonds formed between the complementary bases (of DNA strands) are not
strong enough to hold the strands together. DNA ligase joins (seals) the DNA fragments by
forming a phosphodiester bond between the phosphate group of 5′-carbon of one deoxyribose
with the hydroxyl group 3′-carbon of another deoxyribose (Fig. 6.2).
Phage T4 DNA ligase requires ATP as a cofactor while E.coli DNA ligase is dependent on NAD+. In
each case, the cofactor (ATP or NAD+) is split to form an enzyme—AMP complex that brings
about the formation of phosphodiester bond. The action of DNA ligase is the ultimate step in
the formation of a recombinant DNA molecule.
Alkaline Phosphatase:
Alkaline phosphatase is an enzyme involved in the removal of phosphate groups. This enzyme is
useful to prevent the unwanted ligation of DNA molecules which is a frequent problem
encountered in cloning experiments. When the linear vector plasmid DNA is treated with
alkaline phosphatase, the 5′-terminal phosphate is removed (Fig 6.5). This prevents both re-
circularization and plasmid DNA dimer formation. It is now possible to insert the foreign DNA
through the participation of DNA ligase.
Polymerases:
The groups of enzymes that catalyse the synthesis of nucleic acid molecules are collectively
referred to as polymerases. It is customary to use the name of the nucleic acid template on
which the polymerase acts (Fig. 6.6B). The three important polymerases are given below.
a. DNA-dependent DNA polymerase that copies DNA from DNA.
b. RNA-dependent DNA polymerase (reverse transcriptase) that synthesizes DNA from RNA.
c. DNA-dependent RNA polymerase that produces RNA from DNA
Klenow enzyme
Large (Klenow) Fragment is a proteolytic product of E. coli DNA Polymerase I which retains
polymerization and 3'→ 5' exonuclease activity, but has lost 5'→ 3' exonuclease activity.
Klenow retains the polymerization fidelity of the holoenzyme without degrading 5' termini.
CLONING VECTORS
Plasmids:
Plasmids are extra chromosomal, double- stranded, circular, self-replicating DNA molecules.
Almost all the bacteria have plasmids containing a low copy number (1-4 per cell) or a high copy
number (10-100 per cell). The size of the plasmids varies from 1 to 500 kb. Usually, plasmids
contribute to about 0.5 to 5.0% of the total DNA of bacteria (Note: A few bacteria contain linear
plasmids e.g. Streptomyces sp, Borella burgdorferi).
Types of plasmids:
There are many ways of grouping plasmids. They are categorized as conjugative if they carry a
set of transfer genes (tra genes) that facilitates bacterial conjugation, and non-conjugative, if
they do not possess such genes. Another classification is based on the copy number. Stringent
plasmids are present in a limited number (1-2 per cell) while relaxed plasmids occur in large
number in each cell.
F-plasmids possess genes for their own transfer from one cell to another, while R-plasmids
carry genes resistance to antibiotics. In general, the conjugative plasmids are large, show
stringent control of DNA replication, and are present in low numbers. On the other hand, non-
conjugative plasmids are small, show relaxed control of DNA replication, and are present in high
numbers.
Nomenclature of plasmids:
It is a common practice to designate plasmid by a lowercase p, followed by the first letter(s) of
researcher(s) names and the numerical number given by the workers. Thus, pBR322 is a
plasmid discovered by Bolivar and Rodriguez who designated it as 322. Some plasmids are given
names of the places where they are discovered e.g. pUC is plasmid from University of California.
pBR322 — the most common plasmid vector:
pBR322 of E.coli is the most popular and widely used plasmid vector, and is appropriately
regarded as the parent or grandparent of several other vectors. pBR322 has a DNA sequence of
4,361 bp. It carries genes resistance for ampicillin (Ampr) and tetracycline (TeIr) that serve as
markers for the identification of clones carrying plasmids. The plasmid has unique recognition
sites for the action of restriction endonucleases such as EcoRI, Hindlll, BamHI, Sail and Pstll (Fig.
6.8).
Other plasmid cloning vectors:
The other plasmids employed as cloning vectors include pUC19 (2,686 bp, with ampicillin
resistance gene), and derivatives of pBR322- pBR325, pBR328 and pBR329.
Bacteriophages:
Bacteriophages or simply phages are the viruses that replicate within the bacteria. In case of
certain phages, their DNA gets incorporated into the bacterial chromosome and remains there
permanently. Phage vectors can accept short fragments of foreign DNA into their genomes. The
advantage with phages is that they can take up larger DNA segments than plasmids. Hence
phage vectors are preferred for working with genomes of human cells.
Bacteriophage λ:
Bacteriophage lambda (or simply phage λ), a virus of E.coli, has been most thoroughly studied
and developed as a vector. In order to understand how bacteriophage functions as a vector, it is
desirable to know its structure and life cycle (Fig. 6.9).
Phage λ consists of a head and a tail (both being proteins) and its shape is comparable to a
miniature hypodermic syringe. The DNA, located in the head, is a linear molecule of about 50
kb. At each end of the DNA, there are single-stranded extensions of 12 base length each, which
have cohesive (cos) ends.
On attachment with tail to E.coli, phage X injects its DNA into the cell. Inside E.coli, the phage
linear DNA cyclizes and gets ligated through cos ends to form a circular DNA. The phage DNA
has two fates-lytic cycle and lysogenic cycle.
Lytic cycle:
The circular DNA replicates and it also directs the synthesis of many proteins necessary for the
head, tail etc., of the phage. The circular DNA is then cleaved (to form cos ends) and packed
into the head of the phage. About 100 phage particles are produced within 20 minutes after the
entry of phage into E.coli.
The host cell is then subjected to lysis and the phages are released. Each progeny phage particle
can infect a bacterial cell, and produce several hundreds of phages. It is estimated that by
repeating the lytic cycle four times, a single phage can cause the death of more than one billion
bacterial cells.
If a foreign DNA is spliced into phage DNA, without causing harm to phage genes, the phage will
reproduce (replicate the foreign DNA) when it infects bacterial cell. This has been exploited in
phage vector employed cloning techniques.
Lysogenic cycle:
In this case, the phage DNA (instead of independently replicating) becomes integrated into the
E.coli chromosome and replicates along with the host genome. No phage particles are
synthesized in this pathway.
Use of phage λ as a vector:
Only about 50% of phage λ DNA is necessary for its multiplication and other functions. Thus, as
much as 50% (i.e. up to 25kb) of the phage DNA can be replaced by a donor DNA for use in
cloning experiments. However, several restriction sites are present on phage λ which is not by
itself a suitable vector. The λ-based phage vectors are modifications of the natural phage with
much reduced number of restriction sites.
Insertion vectors:
They have just one unique cleavage site, which can be cleaved, and a foreign DNA ligated in. It
is essential that sufficient DNA (about 25%) has to be deleted from the vector to make space for
the foreign DNA (about 18kb).
Replacement vectors:
These vectors have a pair of restriction sites to remove the non-essential DNA (stuffer DNA)
that will be replaced by a foreign DNA. Replacement vectors can accommodate up to 24kb, and
propagate them. Many phage vector derivations (insertion/ replacement) have been produced
by researchers for use in recombinant DNA technology.
The main advantage of using phage vectors is that the foreign DNA can be packed into the
phage {in vitro packaging), the latter in turn can be injected into the host cell very effectively
(Note: No transformation is required).
Cosmids:
Cosmids are the vectors possessing the characteristics of both plasmid and bacteriophage λ.
Cosmids can be constructed by adding a fragment of phage λ DNA including cos site, to
plasmids. A foreign DNA (about 40 kb) can be inserted into cosmid DNA.
The recombinant DNA so formed can be packed as phages and injected into E.coli (Fig. 6.10).
Once inside the host cell, cosmids behave just like plasmids and replicate. The advantage with
cosmids is that they can carry larger fragments of foreign DNA compared to plasmids.
Artificial Chromosome Vectors:
Human artificial chromosome (HAC):
Developed in 1997 (by H. Willard), human artificial chromosome is a synthetically produced
vector DNA, possessing the characteristics of human chromosome. HAC may be considered as a
self-replicating micro-chromosome with a size ranging from 1/10th to 1/5th of a human
chromosome. The advantage with HAC is that it can carry human genes that are too long.
Further, HAC can carry genes to be introduced into the cells in gene therapy.
Bacterial artificial chromosomes (BACs):
The construction of BACs is based on one F-plasmid which is larger than the other plasmids
used as cloning vectors. BACs can accept DNA inserts of around 300 kb. The advantage with
bacterial artificial chromosome is that the instability problems of YACs can be avoided. In fact, a
major part of the sequencing of human genome has been accomplished by using a library of
BAC recombinant.
Yeast artificial chromosomes (YACs):
Introduced in 1987 (by M. Olson), yeast artificial chromosome (YAC) is a synthetic DNA that can
accept large fragments of foreign DNA (particularly human DNA). It is thus possible to clone
large DNA pieces by using YAC. YACs are the most sophisticated yeast vectors, and represent
the largest capacity vectors available. They possess centromeric and telomeric regions, and
therefore the recombinant DNA can be maintained like a yeast chromosome.
Shuttle Vectors:
The plasmid vectors that are specifically designed to replicate in two different hosts (say in
E.coli and Streptomyces sp) are referred to as shuttle vectors. The origins of replication for two
hosts are combined in one plasmid. Therefore, any foreign DNA fragment introduced into the
vector can be expressed in either host. Further, shuttle vectors can be grown in one host and
then shifted to another host (hence the name shuttle). A good number of eukaryotic vectors
are shuttle vectors.
HOST CELLS – ADVANTAGES AND LIMITATIONS
The hosts are the living systems or cells in which the carrier of recombinant DNA molecule or
vector can be propagated. There are different types of host cells-prokaryotic (bacteria) and
eukaryotic (fungi, animals and plants). Some examples of host cells used in genetic engineering
are given in Table 6.4.
Host cells, besides effectively incorporating the vector’s genetic material, must be conveniently
cultivated in the laboratory to collect the products. In general, microorganisms are preferred as
host cells, since they multiply faster compared to cells of higher organism (plants or animals).
Prokaryotic Hosts:
Escherichia coli:
The bacterium, Escherichia coli was the first organism used in the DNA technology experiments
and continues to be the host of choice by many workers. Undoubtedly, E.coli, the simplest
Gram negative bacterium (a common bacterium of human and animal intestine), has played a
key role in the development of present day biotechnology.
Under suitable environment, E.coli can double in number every 20 minutes. Thus, as the
bacteria multiply, their plasmids (along with foreign DNA) also multiply to produce millions of
copies, referred to as colony or in short clone. The term clone is broadly used to a mass of cells,
organisms or genes that are produced by multiplication of a single cell, organism or gene.
Limitations of E. coli:
There are certain limitations in using E.coli as a host. These include- causation of diarrhea by
some strains, formation of endotoxins that are toxic, and a low export ability of proteins from
the cell. Another major drawback is that E.coli (or even other prokaryotic organisms) cannot
perform post-translational modifications.
Bacillus subtilis:
Bacillus subtilis is a rod shaped non-pathogenic bacterium. It has been used as a host in industry
for the production of enzymes, antibiotics, insecticides etc. Some workers consider B.subtilis as
an alternative to E.coli.
Eukaryotic Hosts:
Eukaryotic organisms are preferred to produce human proteins since these hosts with complex
structure (with distinct organelles) are more suitable to synthesize complex proteins. The most
commonly used eukaryotic organism is the yeast, Saccharomyces cerevisiae. It is a non-
pathogenic organism routinely used in brewing and baking industry. Certain fungi have also
been used in gene cloning experiments.
Mammalian cells:
Despite the practical difficulties to work with and high cost factor, mammalian cells (such as
mouse cells) are also employed as hosts. The advantage is that certain complex proteins which
cannot be synthesized by bacteria can be produced by mammalian cells e.g. tissue plasminogen
activator. This is mainly because the mammalian cells possess the machinery to modify the
protein to its final form (post-translational modifications).
It may be noted here that the gene manipulation experiments in higher animals and plants are
usually carried out to alter the genetic makeup of the organism to create transgenic animals
and transgenic plants rather than to isolate genes for producing specific proteins.
ISOLATION AND PURIFICATION OF NUCLEIC ACIDS (GENOMIC/PLASMID DNA
AND RNA)
Every gene manipulation procedure requires genetic material like DNA and RNA. Nucleic acids
occur naturally in association with proteins and lipoprotein organelles. The dissociation of a
nucleoprotein into nucleic acid and protein moieties and their subsequent separation, are the
essential steps in the isolation of all species of nucleic acids.
Isolation of nucleic acids is followed by quantitation of nucleic acids generally done by either
spectrophotometric or by using fluorescent dyes to determine the average concentrations and
purity of DNA or RNA present in a mixture.
Isolating the genetic material (DNA) from cells (bacterial, viral, plant or animal) involves three
basic steps-
• Rupturing of cell membrane to release the cellular components and DNA
• Separation of the nucleic acids from other cellular components
• Purification of nucleic acids
Isolation and Purification of Genomic DNA
Genomic DNA is found in the nucleus of all living cells with the structure of double stranded
DNA remaining unchanged (helical ribbon). The isolation of genomic DNA differs in animals and
plant cells. DNA isolation from plant cells is difficult due to the presence of cell wall, as
compared to animal cells. The amount and purity of extracted DNA depends on the nature of
the cell.
The method of isolation of genomic DNA from a bacterium comprises following steps –
1. Bacterial culture growth and harvest.
2. Cell wall rupture and cell extract preparation.
3. DNA Purification from the cell extract.
4. Concentration of DNA solution.
Growth and harvest of bacterial culture
Bacterial cell culture is more convenient than any other microbe, as it requires only liquid
medium (broth) containing essential nutrients at optimal concentrations, for the growth and
division of bacterial cells. The bacterial cells are usually grown on a complex medium like Luria-
Bertani (LB), in which the medium composition is difficult to decipher. Later, the cells are
separated by centrifugation and resuspended in 1% or less of the initial culture volume.
Preparation of cell extract
Bacterial cell is surrounded by an additional layer called cell wall, apart from plasma membrane
with some species of E. coli comprising multilayered cell wall. The lysis of cell wall to release the
genetic material i.e. DNA can be achieved by following ways-
• Physical method by mechanical forces.
• Chemical method by metal chelating agents i.e. EDTA and surfactant i.e. SDS or enzyme (e.g.
lysozyme). Lysozyme • present in egg-white, salivary secretion and tears. • catalyzes the
breakdown of cell wall i.e. the peptidoglycan layer. EDTA (Ethylene diamine tetra-acetic acid) •
a chelating agent necessary for destabilizing the integrity of cell wall. • inhibits the cellular
enzymes that degrade DNA. SDS (Sodium dodecyl sulphate) • helps in removal of lipid
molecules and denaturation of membrane proteins. Generally, a mixture of EDTA and lysozyme
is used. Cell lysis is followed by centrifugation to pellet down the cell wall fractions leaving a
clear supernatant containing cell extract.
Purification of DNA
In addition to DNA, a cell extract contains significant quantities of protein and RNA which can
be further purified by following methods.
Organic extraction and enzymatic digestion for the removal of contaminants
It involves the addition of a mixture of phenol and chloroform (1:1) to the cell lysate for protein
separation. The proteins aggregate as a white mass in between the aqueous phase containing
DNA and RNA, and the organic layer. Treatment of lysate with pronase or protease, in addition
to phenol/chloroform, ensures complete removal of proteins from the extract. The RNA can be
effectively removed by using Ribonuclease, an enzyme which rapidly degrades RNA into its
ribonucleotide subunits. Repeated phenol extraction is not desirable, as it damages the DNA. 4-
1.2.3.2. Using ion-exchange chromatography This involves the separation of ions and polar
molecules (proteins, small nucleotides and amino acids) based on their charge. DNA carrying
negative charge binds to the cationic resin or matrix which can be eluted from the column by
salt gradient. Gradual increase in salt concentration detaches molecules from the resin one
after another. Figure 4-1.2. Preparation of genomic DNA
Concentration of DNA samples
Concentration of DNA can be done using ethanol along with salts such as sodium acetate,
potassium acetate etc. These salts provide metal ions like sodium ions (Na+), potassium ions
(K+) which help in aggregation and hence precipitation of DNA molecules. Advantage It leaves
short-chain and monomeric nucleic acid components in solution. Ribonucleotides produced by
the ribonuclease treatment are separated from DNA. 4-1.3. Isolation and Purification of Plasmid
DNA Plasmids are circular, double stranded extra cellular DNA molecules of bacterium and most
commonly used in recombinant DNA technology. The isolation of plasmid DNA involves three
major steps1. Growth of the bacterial cell. 2. Harvesting and lysis of the bacteria. 3. Purification
of the plasmid DNA. 4-1.3.1. Growth of the bacterial cell It involves growth of the bacterial cells
in a media containing essential nutrients. 4-1.3.2. Harvest and lysis of bacteria Lysis of bacteria
results in the precipitation of DNA and cellular proteins. Addition of acetate-containing
neutralization buffer results in the precipitation of large and less supercoiled chromosomal DNA
and proteins leaving the small bacterial DNA plasmids in solution. 4-1.3.3. Purification of
Plasmid DNA This step is same for both plasmid and genomic but former involves an additional
step i.e. the separation of plasmid DNA from the large bacterial chromosomal DNA..
Methods for separation of plasmid DNA
Separation of plasmid DNA is based on the several features like size and conformation of
plasmid DNA and bacterial DNA. Plasmids are much smaller than the bacterial main
chromosomes, the largest plasmids being only 8% of the size of the E. coli chromosome. The
separation of small molecules (i.e. plasmids) from larger ones (i.e. bacterial chromosome) is
based on the fact that plasmids and the bacterial chromosomes are circular but bacterial
chromosomes break into linear fragments during the preparation of the cell extract resulting in
separation of pure plasmids. The methods of separation of plasmid DNA are described as
below4-1.3.3.1.1. Separation based on size difference • It involves lysis of cells with lysozyme
and EDTA (function as described above in point 4-1.2.2.) in the presence of sucrose (prevents
the immediate bursting of cell). • Cells with partially degraded cell walls are formed that retain
an intact cytoplasmic membrane called as sphaeroplasts. • Cell lysis is then induced by the
addition of a non-ionic detergent (e.g. Triton X100) or ionic detergents (e.g. SDS) causing
chromosomal breakage. • Bacterial chromosome attached to cell membrane, upon lysis gets
removed with the cell debris. • A cleared lysate consisting almost entirely of plasmid DNA is
formed with very little breakage of the bacterial DNA. (Figure 4-1.3.3.1.1.).
Separation of plasmid DNA on the basis of size.
Separation based on conformation
Plasmids are supercoiled molecules formed by partial unwinding of double helix of the plasmid
DNA during the plasmid replication process by enzymes called topoisomerases. The supercoiled
conformation can be maintained when both polynucleotide strands are intact, hence called
covalently closed-circular (ccc) DNA. If one of the polynucleotide strands is broken, the double
helix reverts to its normal relaxed state taking an alternative conformation, called open-circular
(oc). Super coiling is important in plasmid preparation due to the easy separation of supercoiled
molecules from non-supercoiled ones.
The commonly used methods of separation based on conformation are as follows4-1.3.3.1.2
(a). Alkaline denaturation method
• This method is based on maintaining a very narrow pH range for the denaturation of non-
supercoiled DNA but not the supercoiled plasmid (Figure 4-1.3.3.1.2(a).). • Addition of sodium
hydroxide to cell extract or cleared lysate (pH12.0-12.5) results in disruption of the hydrogen
bonds of non-supercoiled DNA molecules. • As a result, the double helix unwinds and two
polynucleotide chains separate. • Further addition of acid causes the aggregation of these
denatured bacterial DNA strands into a tangled mass which can be pelleted by centrifugation,
leaving plasmid DNA in the supernatant. Advantage • Most of the RNA and protein under
defined conditions (specifically cell lysis by SDS and neutralization with sodium acetate) can be
removed by the centrifugation. • No requirement of organic extraction.
Separation of plasmid DNA by Alkaline denaturation method 4-1.3.3.1.2(b). Ethidium bromide-
cesium chloride density gradient centrifugation • Density gradient centrifugation can separate
DNA, RNA and protein. It is a very efficient method for obtaining pure plasmid DNA. • A density
gradient is produced by centrifuging a solution of cesium chloride at a very high speed which
pulls the CsCl ions towards the bottom. This process is referred as isopycnic centrifugation. •
The DNA migrates to the point at which it has density similar to that of CsCl i.e.1.7 g/cm3 in the
gradient. • In contrast, protein molecules having lower buoyant densities float at the top of the
tube whereas RNA gets pelleted at the bottom. Density gradient centrifugation in the presence
of ethidium bromide (EtBr) can be used to separate supercoiled DNA from non-super coiled
molecules. Ethidium bromide is an intercalating dye that binds to DNA molecules causing partial
unwinding of the double helix. Supercoiled DNA have very little freedom to unwind due to
absence of free ends and bind to a limited amount of EtBr resulting in very less decrease in
buoyant density (0.085 g/cm3 ) than that of linear DNA (0.125 g/cm3 ). As a result, they form a
distinct band separated from the linear bacterial DNA. The EtBr bound to DNA is then extracted
by n-butanol and the CsCl is removed by dialysis. 4-1.4.
Isolation and Purification of RNA
RNA (Ribonucleic acid) is a polymeric substance consisting of a long single-stranded chain of
phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine and uracil
bonded to the ribose sugar present in living cells and many viruses. The steps for preparation of
RNA involve homogenization, phase separation, RNA precipitation, washing and re-dissolving
RNA. The method for isolation and purification of RNA are as follows
1) Organic extraction method
2) Filter-based, spin basket formats
3) Magnetic particle methods
4) Direct lysis method.
Organic extraction method This method involves phase separation by addition and
centrifugation of a mixture of a solution containing phenol, chloroform and a chaotropic agent
(guanidinium thiocyanate) and aqueous sample. Guanidium thiocyanate results in the
denaturation of proteins and RNases, separating rRNA from ribosomes. Addition of chloroform
forms a colorless upper aqueous phase containing RNA, an interphase containing DNA and a
lower phenol-chloroform phase containing protein. RNA is collected from the upper aqueous
phase by alcohol (2-propanol or ethanol) precipitation followed by rehydration.
METHODS OF GENE TRANSFER:
The six methods are: (1) Transformation (2) Electroporation (3) Liposome-Mediated Gene
Transfer (4) Transduction and (5) Direct Transfer of DNA.6) Particle bombardment
1. Transformation:
Transformation is the method of introducing foreign DNA into bacterial cells (e.g. E.coli). The
uptake of plasmid DNA by E.coli is carried out in ice-cold CaCl2 (0-5°C), and a subsequent heat
shock (37-45°C for about 90 sec). By this technique, the transformation frequency, which refers
to the fraction of cell population that can be transferred, is reasonably good e.g. approximately
one cell for 1000 (10-3) cells.
Transformation efficiency:
It refers to the number of trans-formants per microgram of added DNA. For E.coli,
transformation by plasmid, the transformation efficiency is about 107 to 108 cells per
microgram of intact plasmid DNA. The bacterial cells that can take up DNA are considered as
competent. The competence can be enhanced by altering growth conditions.
The mechanism of the transformation process is not fully understood. It is believed that the
CaCI2 affects the cell wall, breaks at localized regions, and is also responsible for binding of DNA
to cell surface. A brief heat shock (i.e. the sudden increase in temperature from 5°C to 40°C)
stimulates DNA uptake. In general, large-sized DNAs are less efficient in transforming.
Other chemical methods for transformation:
Calcium phosphate (in place of CaCI2) is preferred for the transfer of DNA into cultured cells.
Sometimes, calcium phosphate may result in precipitate and toxicity to the cells. Some workers
use diethyl amino ethyl dextran (DEAE -dextran) for DNA transfer.
2. Electroporation:
Electroporation is based on the principle that high voltage electric pulses can induce cell plasma
membranes to fuse. Thus, electroporation is a technique involving electric field-mediated
membrane permeabilization. Electric shocks can also induce cellular uptake of exogenous DNA
(believed to be via the pores formed by electric pulses) from the suspending solution.
Electroporation is a simple and rapid technique for introducing genes into the cells from various
organisms (microorganisms, plants and animals).
The basic technique of electroporation for transferring genes into mammalian cells is depicted
in Fig. 6.11. The cells are placed in a solution containing DNA and subjected to electrical shocks
to cause holes in the membranes. The foreign DNA fragments enter through the holes into the
cytoplasm and then to nucleus.
Electroporation is an effective way to transform E.coli cells containing plasmids with insert
DNAs longer than 100 kb. The transformation efficiency is around 109 transformants per
microgram of DNA for small plasmids (about 3kb) and about 106 for large plasmids (about 130
kb).
3. Liposome-Mediated Gene Transfer:
Liposomes are circular lipid molecules, which have an aqueous interior that can carry nucleic
acids. Several techniques have been developed to encapsulate DNA in liposomes. The
liposome- mediated gene transfer, referred to as lipofection, is depicted in Fig. 6.12.
On treatment of DNA fragment with liposomes, the DNA pieces get encapsulated inside
liposomes. These liposomes can adher to cell membranes and fuse with them to transfer DNA
fragments. Thus, the DNA enters the cell and then to the nucleus. The positively charged
liposomes very efficiently complex with DNA, bind to cells and transfer DNA rapidly.
Lipofection is a very efficient technique and is used for the transfer of genes to bacterial, animal
and plant cells. T
4. Transduction:
Sometimes, the foreign DNA can be packed inside animal viruses. These viruses can naturally
infect the cells and introduce the DNA into host cells. The transfer of DNA by this approach is
referred to as transduction.
5. Direct Transfer of DNA:
It is possible to directly transfer the DNA into the cell nucleus. Microinjection and particle
bombardment are the two techniques commonly used for this purpose.
Microinjection:
DNA transfer by microinjection is generally used for the cultured cells. This technique is also
useful to introduce DNA into large cells such as oocytes, eggs and the cells of early embryos.
The term transfection is used for the transfer DNA into eukaryotic cells, by various physical or
chemical means.
6.Transfection by particle bombardment
Particle bombardment (also known as biolistics or microprojectile transfection) procedure
involves coating micrometer-sized gold or tungsten particles with DNA and then accelerating
the particles into cells or tissues. A major advantage of this method is that DNA can be
delivered to deep cells in tissue slices, and the depth of penetration can be adjusted by
changing the applied force. The size and total mass of the particles and the force of the
bombardment are important parameters that balance efficient penetration against cell
damage. The technique was developed for the transformation of maize and is now a method of
choice for generating transgenic cereal plants. For animal cells, the technique has been less
widely used because it is usually simpler to transfect cultured cells by alternative well-
established methods. However, the technique has found a role in the transfection of whole
organs and tissue slices, and more recently for the transfer of DNA to surface organs in gene
therapy.