CHAPTER PLANS.NO. PARTICULARS PAGE NO.

1 INTRODUCTION

2 HISTORY

3 TOOLS USED IN RDNA TECHNOLOGY

4 ENZYMES

5 CLONING METHOD

6 COLONY HYBRIDIZATION

7 PCR

8 TRANSGENICS

9 CONCLUSION

10 REFERENCES

1.INTRODUCTIO

N

Recombinant DNA technology refers to the set of techniques for recombining genes from

different sources in vitro and transferring this recombinant DNA into cells where it may be

expressed.

• These techniques were first developed around 1975 for basic research in bacterial

molecular biology, but this technology has also lead to many important discoveries in basic

eukaryotic molecular biology.

• Such discoveries resulted in the appearance of the biotechnology industry.

Biotechnology refers to the use of living organisms or their components to do practical tasks

such as:

The use of microorganisms to make wine and cheese

Selective breeding of livestock and crops

Production of antibiotics from microorganisms

Production of monoclonal antibodies

The use of recombinant DNA techniques allows modern biotechnology to be a more precise

and systematic process than earlier research methods.

It is also a powerful tool since it allows genes to be moved across the species

barrier.

Using these techniques, scientists have advanced our understanding of eukaryotic

molecular biology.

The Human Genome Project is an important application of this technology. This

project’s goal is to transcribe and translate the entire human genome in order to better

understand the human organism.

A variety of applications are possible for this technology, and the practical goal is the

improvement of human health and food production.

2.HISTORY

DNA technology makes it possible to clone genes for basic research and commercial

applications: an overview

Prior to the discovery of recombinant DNA techniques, procedures for altering the genes of

organisms were constrained by the need to find and propagate desirable mutants

Geneticists relied on either natural processes, mutagenic radiation, or chemicals to induce

mutations.

In a laborious process, each organism’s phenotype was checked to determine the presence

of the desired mutation.

Microbial geneticists developed techniques for screening mutants. For example bacteria

was cultured on media containing an antibiotic to isolate mutants which were antibiotic

resistant.

Before 1975, transferring genes between organisms was accomplished by cumbersome and

nonspecific breeding procedures. The only exception to this was the use of bacteria and their

phages.

Genes can be transferred from one bacterial strain to another by the natural processes of

transformation, conjugation and transduction.

Geneticists used these processes to carry out detailed molecular studies on the structure

and functioning of prokaryotic and phage genes.

Bacteria and phages are ideal for laboratory experiments because they are relatively

small, have simple genomes, and are easily propagated.

Although the technique was available to grow plant and animals cells in culture, the

workings of their genomes could not be examined using existing methods.

Recombinant DNA technology now makes it possible for scientists to examine the structure

and function of the eukaryotic genome, because it contains several key components:

Biochemical tools that allow construction of recombinant DNA.

Methods for purifying DNA molecules and proteins of interest.

Vectors for carrying recombinant DNA into cells and replicating it

Techniques for determining nucleotide sequences of DNA molecules.

3.TOOLS USED IN rDNA TECHNOLOGY

3.1.CLONING VECTORS

a) PLASMIDS

b) BACTERIOPHAGE λ

c) COSMID

d) PHASMID

e) SHUTTLE VECTOR

f) YAC/BAC/MAC

g) EXPRESSION VECTORS

3.2ENZYMES

a) RESTRICTION ENZYMES

b) LIGASES

CLONING VECTORS

A cloning vector is a small piece of DNA into which a foreign DNA fragment can be

inserted. The insertion of the fragment into the cloning vector is carried out by treating the

vehicle and the foreign DNA with a restriction enzyme that creates the same overhang,

then ligating the fragments together. There are many types of cloning vectors. Genetically

engineered plasmids and bacteriophages (such as phage λ) are perhaps most commonly used

for this purpose. Other types of cloning vectors include bacterial artificial

chromosomes (BACs) and yeast artificial chromosomes (YACs).

Most commercial cloning vectors have key features that have made their use in molecular

biology so widespread.

In the case of expression vectors, the main purpose of these vehicles is the controlled

expression of a particular gene inside a convenient host organism (e.g. E. coli).

Control of expression can be very important; it is usually desirable to insert the

target DNA into a site that is under the control of a particular promoter. Some

commonly used promoters are T7 promoters, lac promoters (bla promoter)

and cauliflower mosaic virus's 35s promoter (for plant vectors).

To allow for convenient and favorable insertions, most cloning vectors have had

nearly all their restriction sites engineered out of them and a synthetic multiple

cloning site (MCS) inserted that contains many restriction sites. MCSs allow for

insertions of DNA into the vector to be targeted and possibly directed in a chosen

orientation. A selectable marker, such as an antibiotic resistance [e.g. beta-

lactamase (see figure)] is often carried by the vector to allow the selection of

positively transformed cells (see Screening below). All plasmids must carry a

functional origin of replication (ORI; not shown in figure).

Some other possible features present in cloning vectors are: vir genes for plant

transformation, integrase sites for chromosomal insertion, lacZα fragment for α

complementation and blue-white selection, and/or reporter genes in frame with and

flanking the MCS to facilitate the production of recombinant proteins [e.g. fused to

the Green fluorescent protein (GFP) or to the glutathione S-transferase (see figure)].

Now, the different cloning vectors are:-

a) PLASMIDS

Plasmids are molecules of DNA that are found in bacteria separate from the bacterial

chromosome.

They are small (a few thousand base pairs)

They are usually carry only one or a few genes

They are circular

have a single origin of replication.

Plasmids are replicated by the same machinery that replicates the bacterial chromosome.

Some plasmids are copied at about the same rate as the chromosome, so a single cell is apt to

have only a single copy of the plasmid. Other plasmids are copied at a high rate and a single

cell may have 50 or more of them.

Genes on plasmids with high numbers of copies are usually expressed at high levels. In

nature, these genes often encode proteins (e.g., enzymes) that protect the bacterium from one

or more antibiotics.

Plasmids enter the bacterial cell with relative ease. This occurs in nature and may account for

the rapid spread of antibiotic resistance in hospitals and elsewhere. Plasmids can be

deliberately introduced into bacteria in the laboratory transforming the cell with the incoming

genes.

Desirable properties of plasmids

Plasmids have proven extremely useful as cloning vectors, particularly when they are

modified specifically for that purpose. A bacterial plasmid that is designed for cloning should

have some or all of the following properties:

It should be small.

The aim of most cloning experiments is to isolate the passenger DNA. The vector

only serves to carry and amplify the passenger. A small plasmid has the advantage of

contributing only a minimal amount of extraneous DNA to the plasmid/passenger

construct, thereby making it easier to prepare large amounts of the passenger DNA. In

addition, it is easier to get smaller pieces of DNA into bacteria than larger ones --

everything else being equal -- and the smaller the plasmid is, the less it contributes to

total size. Another point is that small plasmids replicate faster and require less energy

for replication than large ones. Finally, small plasmids are easier to purify than large

ones because they are less fragile.

Its DNA sequence should be known.

Knowledge of the DNA sequence of a plasmid allows the genetic engineer to

manipulate it with the full complement of recombinant DNA techniques.

It should grow to high copy number in the host cell.

In other words, relaxed replication plasmids are most often preferred to those that are

under stringent control. In most cases, having many copies makes it easier to purify the

plasmid away from the chromosomal DNA, and of course increases its yield. However, there

are circumstances when just one or a few copies of a plasmid are desirable.

It should contain a selectable marker that allows cells containing the plasmid to

be isolated.

Bacterial cells that harbor plasmids don't necessarily look or act different than those

that do not. It is only when the plasmid carries a gene that lends a special trait to the

bacteria that the molecular engineer can tell if a bacterium contains a plasmid.

Antibiotic resistance is one such trait or marker, and the genes for ampicillin

resistance and tetracycline resistance are common genes carried by frequently used

plasmids. Some plasmids are also readily lost from cells, and unless there is a

selectable gene contributed by the plasmid and the bacteria are kept under selective

conditions, the molecular engineer may end up with a plasmid-less population of cells

at the end of an experiment.

It should also contain a second selectable gene that is inactivated by insertion of

the passenger.

Imagine the following scenario. A scientist is trying to insert a passenger molecule into a

plasmid. The plasmid has only a single selectable marker. After ligation, some of the

molecules contain the passenger. Others do not. These may be plasmids that have

recircularized in the presence of DNA ligase. This mixture of plasmids is then used to

transform bacteria. Both recombinant plasmids and recircularized plasmids contain the

same marker and cannot readily be told apart genetically. How are colonies carrying a

plasmid with a passenger distinguished from those with a plasmid with no passenger?

One way is to grow many individual colonies, isolate plasmid DNA from each, run the

DNA out on an agarose gel, and identify the recombinant plasmid molecules because they

are bigger by virtue of the passenger that they carry. While this procedure works and is

very commonly carried out, it is laborious, time consuming, and expensive.

There is a better way. Suppose that a plasmid carries two different antibiotic

resistance genes, A and B. What would be the consequences of cloning the passenger

into the middle of gene B? In most cases, the foreign DNA will disrupt gene B and

not allow it to work. On the other hand, if the plasmid simply recircularizes, gene B

will be unaffected. With this arrangement it is possible to tell which cells harbor

plasmids that carry passengers. Those that contain any plasmid at all will have gene A

activity. And those that have a plasmid bearing a passenger, will lack activity from

gene B.

There should be a large number of unique restriction sites lying within one of the

two selectable markers described above.

As described above, most cloning is done by inserting a fragment of DNA that is cut

with a specific restriction endonuclease into a vector that is cut with the same enzyme

(remember, this ensures that the two have compatible ends). If the vector contains

more than one such site, it will be cut into multiple pieces by the restriction enzyme,

complicating matters unnecessarily. The presence of many unique sites allows for

maximum flexibility and ease in cloning.

Plasmid purification

Plasmids may be purified from bacteria by taking advantage of the difference between the

small, circular plasmid molecules and the large, broken (hence linear) pieces of chromosomal

DNA. (Chromosomal DNA from E. coli is also circular, but during isolation its relatively

large size (six million nucleotide pairs) and consequent fragility cause it to break easily.)

The most common method for purifying plasmid DNA involves three steps:-

First, the bacteria are broken open and the is DNA isolated.

Then the DNA is denatured.

Finally, the DNA is renatured and centrifuged.

Upon denaturation, the two circular single-stranded chains of the plasmid DNA remain

entwined and don't separate fully. When conditions are set up so that renaturation can occur,

each strand rapidly finds its complement. The chromosomal DNA, on the other hand, breaks

readily and therefore consists of noncircular pieces. Under these circumstances, the two

strands easily denature and separate. Upon renaturation, they have difficulty finding complete

copies of their complements. Often partial renaturation occurs between different sizes of

single-stranded fragments and large insoluble aggregates form. These can be simply

separated from the small circular plasmid DNA by high-speed centrifugation.

Some popular plasmids are:-

pAMP

4539 base pairs

a single replication origin

a gene conferring resistance to the antibiotic ampicillin (a relative of penicillin)

a single occurrence of the sequence

a fragment of 3755 base pairs carrying both the ampr gene and the replication origin

a fragment of 784 base pairs

both fragments have sticky ends

pBR322

The first really useful plasmid for genetic engineering, pBR322, was pieced together by

Francisco Bolivar, and others in Herbert Boyer's laboratory in the 1970s (The "B" stands for

Bolivar and the "R" for Rodriguez, another scientist in Boyer's laboratory). What makes

pBR322 useful is that it contains an ampicillin resistance gene and a tetracycline resistance

gene. In addition it has a relaxed origin of replication (indicated by the green arrow shown at

the bottom of the illustration) and accumulates to high numbers in E. coli. Its entire 4363

base-pair sequence has been determined, and 21 common enzymes are available that

recognize only a single site within it. (However, only 11 are in either of the two antibiotic

resistance genes.).

pUC18

More recently, a series of small plasmids (about 2.7 kilobase pairs) have been developed in

Joachim Messing'e laboratory that have several properties that have made them very popular

with genetic engineers. These pUC (pronounced PUCK) plasmids, exemplified by pUC18

pictured at the right, carry an ampicillin resistance gene and an origin of replication, both

from pBR322. They also bear a multiple cloning site -- a sequence of DNA that carries many

restriction sites (13, in the case of pUC18). The multiple cloning site of the pUC plasmids is

special because it also codes for a small peptide. This peptide will correct a specific mutation

in the chromosomal gene that codes for the enzyme beta-galactosidase.

When plasmids containing this sequence are cloned into a specific E. coli strain that lacks

beta-galactosidase activity, they make the peptide and thereby begin to express active

enzyme. If, however, one of the restriction endonuclease sites in the multiple cloning site is

opened and a foreign gene inserted, the peptide is no longer produced (because the protein

coding region is disturbed), and no beta-galactosidase activity appears.

Cells that harbor an active beta-galactosidase enzyme can be made to turn blue in the

presence of certain substrates. Those colonies that have a passenger inserted at the multiple

cloning site of the pUC plasmids will lack the enzyme and will be white, while those that

have simply recircularized (those that don't contain a passenger) will stain blue. In this way,

pUC plasmids containing a foreign insert of DNA can be distinguished from plasmids

without a passenger.

b. BACTERIOPHAGE λ

DNA is extracted from phage λ and the central gene cluster is

removed by restriction endonuclease digestion. The foreign

DNA to be cloned is cut with the same enzyme and ligated to

the left and right “arms” of the phage λDNA. The

recombinant DNA is then mixed with phage proteins in vitro.

The DNA is packaged into the phage head and tail fibers are

attached via a self-assembly pathway. The recombinant viral

particle is then able to infect bacterial cells on an agar plate.

The phage replicates its genome, including the foreign DNA

insert.

Recombinant phage λ DNA directs the cell to make phage particles. The bacteria become

filled with new phage particles, break open (lyse), and release millions of recombinant

phages. The holes in the lawn of host bacteria, called plaques, are regions where phages have

killed the bacteria. Each plaque represents progeny of a single recombinant phage.

Figure:-Lysis plaques of lambda phage on E. coli bacteria

Although plasmids make good general purpose cloning vectors, there are limitation to their

use. The major problem relates to the size of insert, which can be effectively carried into the

cell. Transformation frequencies sharply decrease as the size of the DNA circle increases. In

addition, plasmids with large inserts are unstable and give rise to smaller segregants.

Bacteriophage lambda has been widely used in recombinant DNA since engineering of

the first viral cloning vector in 1974. Phage lambda vectors are particularly useful for

preparing genomic libraries, because they can hold a larger piece of DNA than a plasmid

vector. The phage lambda has 2 modes by which it replicates itself- the 1 st is LYTIC CYCLE

which involves attachment of the phage to the bacterial cell, infection of the phage DNA and

the replication of this DNA inside the cell. After the packaging of the DAN molecule in

protein coats, the cell will LYSE, liberating the progeny phage.

The 2nd mode of replication is called as LYSOGENY, follows the

same initial path of infection, but the phage genome, rather than

independently replicating becomes integrated into the bacterial

chromosome. In this case, the cell does not lyse and the phage

genome become a silent passenger (Benign guest OR Prophage).

The phage lambda genome is a 49kb mol packaged in a protein, attached to a tail that

promotes the infection of the phage DNA into the cell. Replacement vectors have paired

cloning sites on either side of a central gene cluster. This central cluster contains genes for

lysogeny and recombination, which are not essential for the lytic life cycle. The central gene

cluster can be removed and foreign DNA inserted between the “arms”. All phage vectors

used as cloning vectors have been disarmed for safety and can only function in special lab

conditions. The recombinant viral particle infects bacterial host cells, in a process called

“transduction”. The host cells lyse after phage reproduction, releasing progeny virus particles.

The viral particles appear as a clear spot of lysed bacteria or “plaque” on an agar plate

containing a lawn of bacteria. Each plaque represents progeny of a single recombinant phage

and contains millions of recombinant phage particles. Most contemporary vectors carry a

lacZ’ gene allowing blue- white selection. In this type, it is possible to accommodate inserts

of up to 24kb and propagate them in a stable fashion. The great advantage of using phage

vectors is that after the foreign DNA has been ligated in the products can be packaged in the

test tube into phage coat. This process is called IN- VITRO PACKAGING- allows the

r-DNA to be added to the cell by phage infection instead of transformation/ transfection, thes

increasing the efficiency by many orders of magnitude.

Phage lambda as a vector-

There are 2 kinds of lambda vectors which differ in size, they are-

a) Lambda replacement vectors-

Contains a restriction site for phage propagation in a suitable bacterial host.

Remaining part of the lambda genome is removed and is replaced by a foreign DNA.

b) Lambda insertion vectors-

When cloning into an insertion vector, the phage DNA is cleaved with a restriction

enzyme that cut its site. No phage DNA is removed, therefore, much lower size of

DNA can be inserted.

Three reasons why bacteriophage lambda is a good cloning vehicle

1. It can accept very large pieces of foreign DNA. About 20kb of DNA can be deleted from

its central region (between the J and N genes) and elsewhere, and replaced with an equal

quantity of foreign DNA without affecting the ability of the phage to grow lytically.

2. It has been extensively reworked over the years. Genetic engineers have constructed

numerous derivatives of lambda that contain only one or two sites for a variety of

restriction enzymes.

3. Finally -- its main advantage -- bacteriophage lambda is one of the few organisms that

can be reconstituted in a test tube. By simply mixing phage DNA with a mixture of phage

proteins, an infective viral particle with the DNA inside the phage head can be produced.

This process is called in vitro packaging and it normally occurs when long tandem arrays

of phage chromosomes (they are produced in this form when they replicate in the

bacterial cell) are cleaved into monomers by certain phage proteins. The DNA is cut at a

site on the chromosome called cos, and this operation produces the cohesive ends that

were mentioned above. Ordinarily the DNA is cut into 48.5 kb fragments that are

incorporated into the head of newly formed phage particles.

c) COSMID

While plasmids and bacteriophage lambda are both highly useful as vectors, they impose two

limitations-

a) The size of the DNA fragments that can be cloned in such vectors is limited- with

plasmids, the larger the inserted fragments of foreign DNA, the lower the efficiency of

ligation and transfection, thus the cloning of DNA fragments larger than 15kb is

experimentally difficult.

In case of lambda vectors, the length of the non-essential region of the lambda DNA limits of

fragment size to 24kb or less (20kb).

b) The original lambda vectors don’t allow propagation of viable bacterial cells that carry the

inserted DNA fragments. The insert is propagated as a part of a virus that lyse the cell.

So Cosmids were developed as vectors specifically designed for the cloning of large

fragments of DNA (45kb). Cosmids combine the properties of plasmids and bacteriophage

lambda vectors. For example, the commonly used cosmid pLFR-5 has two cos sites (cos

ends) from bacteriophage lambda separated by a ScaI restriction endonuclease site, a multiple

cloning site with six unique recognition sites (HindIII, PstI, SalI, BamHI, SmaI and EcoRI),

an origin of DNA replication and a tetracycline resistance (Tetr) gene. The pLFR-5 DNA

(6kb) is cleaved initially with ScaI and then with BamHI. The final 2 DNA samples are

mixed and ligated. Some of the ligated products will have a 45kb DNA piece inserted

between the 2 fragments that are derived from the digestions of the pLFR-5 DNA. These

molecules will be about 50kb long and have cos sequences that are about 50kb apart.

Consequently, these DNA constructs are successfully packaged into bacteriophage lambda

heads in vitro. After assembly of bacteriophage particles, the DNA is delivered by infection

into E.coli, once inside the host cell, the cos ends, which were cleaved during the in vitro

packaging, base pair and enable the linear DNA to circularize. Moreover, the tetracycline

resistance gene allows colonies that carry the cosmid to grow in the presence of tetracycline.

Non- transformed cells are sensitive to tetracycline and die.

d) PHASMIDS

A 2nd combination of plasmid and phage lambda sequence has been devised to exploit the

viruses of each type of vectors. This combination consists of a plasmid vector carrying a

lambda attachment site. The plasmid may insert into a phage lambda genome (by means of

site- specific recombination/ lytic cycle) generating a phage genome containing one or more

plasmid molecule (depending on the length of the plasmid). These novel genetic combination

are called “phasmids”. They contain functional origins of replication of the plasmids and of

lambda and may be propagated as a plasmid or as a phasmid in appropriate E.coli strains.

Phage particles are easy to store, they have an effectively infinite shell- life, screening

phage plaques by molecular hybridization often gives clean results than screening bacterial

colonies.

e) SHUTTLE VECTORS

That can replicate in different organisms. They contain sequences from E.coli plasmid and a

particular region of the yeast genome.

Essential Features are-

a) Two replication origins (origin active in yeast and one in E.coli)

b) Two selective markers (trp detectable in yeast and ampicilin resistance detectable in

E.coli).

c) Restriction sites next to a yeast promoter, such a vector can be cleaved and a yeast

DNA fragment can be inserted but most important, the hybrid vector will transform trp yeast

cells, producing trp+ cells.

f) YAC

Large DNA sequence can be cloned in Yeast Artificial Chromosome (YAC). YACs are linear

DNA segments that contain all the molecular components required for replication in yeast.

YAC contains-

A replication origin called an autonomously replicating sequence (ARE),

A centromere sequence,

The telomeres (the ends of linear chromosome)

They are also called Mini Chromosome. DNAs of several 100kb (200- 400kb) can be

introduced into YACs and successfully cloned.

YAC vectors are maintained as a circle prior to inserting foreign DNA. After cutting with

restriction endonucleases BamHI and EcoRI, the left arm and right arm become linear, with

the end sequences forming the telomeres. Foreign DNA is cleaved with EcoRI and the YAC

arms and foreign DNA are ligated and then transferred into yeast host cells. The yeast host

cells are maintained as spheroplasts (lacking yeast cell wall). Yeast cells are grown on

selective nutrient regeneration plates that lack uracil and tryptophan, to select for molecules

in which the arms are joined bringing together the URA3 and TRP1 genes.

g) EXPRESSION VECTOR

The primary function of a vector in r-DNA technology is to carry a gene of interest into a

recipient cell. When such a transfer is successful, the foreign gene should be expressed in the

recipient cell. Sometimes the foreign gene in the recipient cell may not be expressed. This

may be due to the following reasons-

a) Transcription of the gene didn’t occur due to the absence of an effective promoter.

b) A termination sequence to terminate transcription may be absent.

c) The initiation codon for a particular codon is absent.

Hence, the vectors are constructed in such a way that they contain suitable

expression signals. Such vectors are called Expression vectors. A foreign gene carried by

expression vector into the recipient cell will have complete expression, i.e. the gene will be

transcribed into mRNA and then translated into the protein (gene product).

Such vectors that can produce only the proteins encoded by the foreign gene are

constructed by linking a suitable and strong prokaryotic promoter, a bacterial S-D sequence

and a start codon in front of derived structural gene. This will allow the synthesis of the

corresponding gene product as a pure fused protein.

4.ENZYME

s

Two major categories of enzymes are important tools in the isolation of DNA and the

preparation of recombinant DNA: restriction endonucleases and DNA ligases. Restriction

endonucleases recognize a specific, rather short, nucleotide sequence on a double-stranded

DNA molecule, called a restriction site, and cleave the DNA at this recognition site or

elsewhere, depending on the type of enzyme. DNA ligase joins two pieces of DNA by

forming phosphodiester bonds.

4.1. RESTRICTION ENDONUCLEASE

Restriction enzymes are major tools in recombinant DNA technology.

First discovered in the late 1960s, these enzymes occur naturally in bacteria where they

protect the bacterium against intruding DNA from other organisms.

An "endonuclease" is an enzyme that cuts duplex DNA in the middle, not at an end (for

exonuclease).   Different species of bacteria have evolved different restriction endonucleases,

each to cut foreign DNA that gets into their cells by mistake.  To be cut, the DNA has to lack

their own pattern of protective methylation.   There are well over a hundred restriction

enzymes, each cutting in a very precise way a specific base sequence of the DNA molecule.

A restriction endonuclease cuts DNA only at a specific site, usually containing 4-6 base

pairs.  The enzyme has to cut the DNA backbone twice, recognizing the same type of site;

therefore, the site "reads" the same way backwards as forwards--a palindrome.

Their grouping is based on the types of sequences recognized, the nature of the cut made in

the DNA, and the enzyme structure. Type I and III restriction endonucleases are not useful

for gene cloning because they cleave DNA at sites other than the recognition sites and thus

cause random cleavage patterns. In contrast, type II endonucleases are widely used for

mapping and reconstructing DNA in vitro because they recognize specific sites and cleave

just at these sites In addition, the type II endonuclease and methylase activities are usually

separate, single subunit enzymes.Although the two enzymes recognize the same target

sequence, they can be purified separately from each other. Some type II restriction

endonucleases do not conform to this narrow definition, making it necessary to define further

subdivisions. The discussion here will focus on the “orthodox” type II restriction

endonucleases that are commonly used in molecular biology research.

This "sticky ends" from two different DNA molecules can hybridize together; then the nicks

are sealed using ligase. (Where does ligase come from? What is its natural function?) The

result is recombinant DNA. When this recombinant vector is inserted into E. coli, the cell

will be able to process the instructions to assemble the amino acids for insulin production.

More importantly, the new instructions are passed along to the next generation of E. coli cells

in the process known as gene cloning.

4.2 .DNA LIGASE

The study of DNA replication and repair processes led to the discovery of the DNA-joining

enzyme called DNA ligase. DNA ligases catalyze formation of a phosphodiester bond

between the 5′-phosphate of a nucleotide on one fragment of DNA and the 3′-hydroxyl of

another. This joining of linear DNA fragments together with covalent bonds is called ligation.

Unlike the type II restriction endonucleases, DNA ligase requires ATP as a cofactor. Because

it can join two pieces of DNA, DNA ligase became a key enzyme in genetic engineering. If

restriction-digested fragments of DNA are placed together under appropriate conditions, the

DNA fragments from two sources can anneal to form recombinant molecules by hydrogen

bonding between the complementary base pairs of the sticky ends. However, the two strands

are not covalently bonded by phosphodiester bonds. DNA ligase is required to seal the gaps,

covalently bonding the two strands and regenerating a circular molecule. The DNA ligase

most widely used in the lab is derived from the bacteriophage T4. T4 DNA ligase will also

ligate fragments with blunt ends, but the reaction is less efficient and higher concentrations of

the enzyme are usually required in vitro. To increase the efficiency of the reaction,

researchers often use the enzyme terminal deoxynucleotidyl transferase to modify the blunt

ends. For example, if a single-stranded poly(dA) tail is added to DNA fragments from one

source, and a single stranded poly (dT) tail is added to DNA from another source, the

complementary tails can hydrogen bond.

Recombinant DNA molecules can then be created by ligation.

5.CLONING

MeTHOD

5.1.Genomic Library:

A genomic library contains DNA fragments that represent the entire genome of an organism.

The first step in creating a genomic library is to break the DNA into manageable size pieces

usually by partial restriction endonuclease digest. Under limiting conditions, any particular

restriction site is cleaved only occasionally, so not all sites are cleaved in any particular DNA

molecule. This generates a continuum of overlapping fragments. The second step is to purify

fragments of optimal size by gel electrophoresis or centrifugation techniques. With an

average insert size of 20 kb, the number of random fragments to ensure with high probability

(95–99%) that every sequence is represented is approximately 106 clones for humans.

5.2.cDNA Library:

The principle behind cDNA cloning is that an mRNA population isolated from a specific

tissue, cell type, or developmental stage (e.g. embryo mRNA) should contain mRNAs

specific for any protein expressed in that cell type or during that stage, along with

“housekeeping” mRNAs that encode essential proteins such as the ribosomal proteins, and

other mRNAs common to many cell types or stages of development. Thus, if mRNA can be

isolated, a small subset of all the genes in a genome can be studied. mRNA cannot be cloned

directly, but a cDNA copy of the mRNA can be cloned. Because a cDNA library is derived

from mRNA, the library contains the coding region of expressed genes only, with no introns

or regulatory regions. This latter point becomes important for applications of recombinant

DNA technology to the production of transgenic animals and for human gene therapy

6.COLONY

HYBRIDIZATION

Hybrid molecular complexes (usually called hybrids) of the following types can exist (other

combinations are possible):

DNA-DNA. A single-stranded DNA molecule can form a double-stranded, base-

paired hybrid with a ssDNA target if the probe sequence is the reverse complement of

the target sequence.  A radiolabeled DNA probe can be applied to DNA from a gel

transferred to a membrane, called a Southern Blot (named for its inventor). 

DNA-RNA. A single-stranded DNA (ssDNA) probe molecule can form a double-

stranded, base-paired hybrid with an RNA (RNA is usually a single-strand) target if

the probe sequence is the reverse complement of the target sequence.  An RNA can be

radiolabeled to probe a Southern Blot; or, a ssDNA probe can be applied to

membrane-bound RNA, called a Northern Blot (name is a pun on Southern.) 

Protein-Protein. An antibody probe molecule (antibodies are proteins) can form a

complex with a target protein molecule if the antibody's antigen-binding site can bind

to an epitope (small antigenic region) on the target protein. In this case, the hybrid is

called an 'antigen-antibody complex' or 'complex' for short.  A radiolabeled antibody

can probe membrane-bound proteins, called a Western Blot (an even worse pun.) 

There are two important features of hybridization:

Hybridization reactions are specific - the probes will only bind to targets with

Scomplimentary sequence (or, in the case of antibodies, sites with the correct 3-d

shape). 

Hybridization reactions will occur in the presence of large quantities of molecules similar but

not identical to the target. That is, a probe can find one molecule of target in a mixture of

zillions of related but non-complementary molecules

7.PCR

(POLYMERASE

CHAIN

REACTION)

PCR (POLYMERASE CHAIN REACTION)

In PCR, a heat-stable DNA polymerase is used, most commonly Taq Polymerase from the

thermophilic microbe Thermus aquaticus.  Thomas Brock discovered T. aquaticus from a hot

spring at Yellowstone National Park.

More recently, an even more heat-resistant polymerase has been developed from a

hyperthermophilic microbe growing at 110 degrees C in hydrothermal vent ecosystems in the

deep ocean; it's called "Vent Polymerase." The Taq Polymerase is put with the DNA to be

amplified, plus all four NTPs, plus two primers facing each other, about 200 - 6000 kb

apart.The primers are selected based on the DNA region you want to amplify.  The tube is

placed in a thermal cycler. DNA gets synthesized from each primer, for about 2 minutes. 

Then the temperature is raised to 95 degrees C -- enough to denature (split apart) the DNA

base pairs.  But the Taq Polymerase remains intact, because it comes from an organism that

evolved to grow at this temperature. Now the temperature is decreased again, and primers

again can hybridize to the DNA--both the old AND the newly synthesized strands.  Again,

Taq Polymerase extends new DNA strands.  Again, the temperature is raised. After repeated

cycles, the amount of DNA sequence between the two primers increases exponentially.  First

2 strands, then 4, 8, 16, up to about a million.  Thus, in a couple of hours, you can get

million-fold amplification of a DNA sequence.

PCR has replaced cloning for many purposes, particularly the sequencing of DNA.  It is

faster and requires no vectors, which can mutate as they reproduce.   It can be used

forensically, to amplify tiny amounts of DNA from criminal evidence; or clinically, to detect

DNA sequences linked to inherited disorders.

8.TRANSGENICS

Cloning in Animals.

Animals have two different classes of cells: germ line and somatic. Only alterations in the germ cells can be transmitted to future generations.  However, some forms of somatic cell gene therapy can be useful in treating patients.  For example, people with cystic fibrosis can receive the cloned CFTR gene in a nasal spray, which then infects the lining of their lungs and improves lung function.  The infected (transformed) epithelial cells eventually are lost, however, during normal processes of tissue growth, so the treatment needs to be repeated.

Germ-line cloning.  There are two basic ways to clone a gene in the mammalian germ line.

Inject a DNA fragment containing your gene into the nucleus of a fertilized egg (germ-line gene cloning) or of body tissues in a mature host (somatic gene cloning).  The DNA gets taken up at random somewhere in one of the chromosomes.  An example of germ-line cloning is the injection of angiopoietin cDNA into the fertilized mouse egg. (An example of somatic cloning is gene therapy for cystic fibrosis: inhale a vector containing the CF gene, which gets incorporated into the DNA of cells lining the lungs.  Somatic cloned genes are not inherited by offspring.)  

Put a transgene containing positive and negative selective markers into an embryonic stem cell tissue culture.  The ES cells take up the gene by homologous recombination, replacing the host allele.  The ES cells now are injected into a blastula of an unrelated host, and some of the next generation progeny arise from the ES cells. 

But when you create a clone, you need to know:

DNA: Did the transgene really incorporate into the genome?  Where?  RNA: Is the RNA expressed?  (Or prevented from expression, by a null allele?)  Protein: Is a desired protein expressed

Cloning in Plants.

Plants don't have separate somatic and germ cells, so creating transgenic plants are easier than creating transgenic animals. Techniques include the use of microprojectile bombardment (the "gene gun") and the use of Agrobacterium tumefaciens.