Once the proper DNA fragments have been obtained , they must be joined. This is accomplished by the following methods. When cleavage with a restriction endonuclease creates cohesive ends, these can be annealed with similarly cleaved DNA from another source, including a vector molecule. When such molecules associate, the joint has nicks a few base pairs apart in opposite strands. The enzyme DNA ligase can then repair.

Use of DNA ligase to create a covalent DNA recombinant joined through association of termini generated by Eco R1.

These nicks to form an intact, duplex recombinant molecule, which can be used for transformation and the subsequent selection of cells containing the recombinant molecule. Cohesive ends can also be created by the addition of synthetic DNA linkers to blunt-ended DNA molecules. These linkers are short DNA duplexes containing the recognition site for a restriction enzyme which produces cohesive termini. Linkers are ligated to blunt-ended passenger DNA molecules by DNA ligase encoded by the phage T4. After digesting the product with the restriction enzyme that cleaves the linkers, the products can be ligated to any vehicle DNA via the complementary termini and then cloned.

Homopolymer tailing

Another method for joining DNA molecules involves the addition of Homopolymer extensions to different DNA populations followed by an annealing of complementary Homopolymer sequences.

For example, short nucleotide sequences of pure adenine can be added to the 3’ ends of one population of DNA molecules and short thymine blocks to the 3’ ends of another population. The two types of molecules can then anneal to form mixed dimeric circles that can be used directly for transformation. Single-stranded gaps that may remain in the two strands at each connection will be repaired in the transformed cell.
Blunt-end ligation T4 DNA ligase carries out the intermolecular joining of DNA substrates at completely base-paired ends; such blunt ends can be produced by cleavage with a restriction enzyme or by mechanical shearing followed by enzyme treatment. Transformation The desired DNA sequence, one attached to a DNA vector, must be transferred to a suitable host. Transformation is defined as the introduction of a cell with DNA form a virus is usually referred to as transfection. Transformation in any organism involves (1) a method that allows the introduction of DNA into the cell and (2) the stable integration of DNA into a chromosome, or maintenance of the DNA as a self-replicating entity.

Prokaryotes and lower eukaryotes Escherichia coli is usually the host of choice for cloning. Experiments, and transformation of E. coli is an essential step in these experiments. Escherichia coli treated with calcium chloride are able to take up DNA from bacteriophage lambda as well as plasmid DNA. Calcium chloride is though to effect some structural alterations in the bacterial cell wall. In addition to E. coli, other prokaryotes have also been used as hosts for cloning experiments, among them the nonenteric bacteria, Bacillus species, and actinomycetes.

An efficient method for transformation in Bacillus involves polyethylene glycol-induced DNA uptake in bacterial protoplasts and subsequent regeneration of the bacterial cell wall. Actinomycetes can be similarly transformed. Transformation can also be achieved by first entrapping the DNA with liposomes followed by their fusion with the host cell membrane. Similar transformation methods have been developed for lower eukaryotes such as the yeast Saccharomyces cerevisiae and the filamentous fungus Neurospora crassa. Animal cells Several methods are available of the transfer of DNA into cells of higher eukaryotes.

Specific genes or entire viral genomes can be introduced into cultured mammalian cells in the form of a coprecipitate with calcium phosphate. DNA complexed with calcium diethylamino-ethyl-dextran (DEAE-dextran), or DNA trapped in liposomes or erythrocyte ghosts may also be used in mammalian transformation. Alternatively, bacterial protoplasts containing plasmids can be fused to intact animal cells with the aid of chemical agents such as polyethylene glycol (PEG). Finally, DNA can be directly introduced into cells by microinjection. The efficiency of transfer by each of these methods is quite variable.

Higher plants Introduction of DNA sequences by insertion into the transforming (T)-DNA region of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens is a method of plasmid of Agrobacterium tumefaciens is a method of introducing DNA into plant cells and ensuring its integration. The Ti plasmid has been used to introduce selectable marker genes, including bacterial genes coding for antibiotic resistance, into plant cells. The control sequences used to express these chimeric genes are derived from plant genes. Many foreign genes have been expressed in the transformant plant cells and have conferred selectable properties into plant cells.

Although the Ti plasmid can be used as a gene transfer vector, its use is limited in that A. tumefaciens, the bacterium that transmits Ti plasmids, normally infects primarily dicotyledonous plants. Monocotyledonous plants, including economically important cereasls, cannot be infected by the Ti plasmid. Because of the limitations of the host range of A. tumefaciens, alternative transformation systems are being developed for gene transfer in plants. They include the use of liposomes, as well as induction of DNA uptake in plant protoplasts. With those methods, however, transformation frequencies are low. Plant DNA viruses, such as the cauliflower mosaic virus, might also be used as vectors for DNA transfer.

Foreign DNA has been introduced into plant cells by a technique called electroporation. This technique involves the use of electric pulses to make plant plasma membranes permeable to plasmid DNA molecules. Plasmid DNA taken up in this way has been shown to be stably inherited and expressed. Cloning Vectors There is a large variety of potential vectors for cloned genes. The vectors differ in different classes of organisms. Prokaryotes and lower eukaryotes Three types of vectors have been used in these organisms; plasmids, bacteriophages, and cosmids. Plasmids
Plasmids are extrachromosomal DNA sequences that are stably inherited. Escherichia coli and its plasmids constitute the most versatile type of host-vector system known for DNA cloning.

Several natural plasmids, such as ColE1, have been used as cloning vehicles in E. coli. In addition, a variety of derivatives of natural plasmids have been constructed by combining DNA segments and desirable qualities of older cloning vehicles. These qualities, which facilitate manipulation of cloned DNA and limit the ability of recombinants to survive in the environment beyond the laboratory, include (1) reduced molecular weight of the plasmid; (2) construction of only one site for a given restriction enzyme located in a region not essential for plasmid replication; (3) introduction of one or more phenotypic markers into the plasmid to permit selection of cells containing the plasmid and to aid in the identification of plasmids that have incorporated the foreign DNA.

It is also desirable to use plasmids that can be obtained in high yields. The most versatile and widely used of these plasmids is pBR322. This plasmid has been completely sequenced, providing a detailed restriction enzyme cleavage map and DNA sequence information. The pBR322 plasmid carries the genes coding for resistance to the antibiotics ampicillin and tetracycline.

Transformation in yeast has been demonstrated using a number of plasmids, including vectors derived from the naturally occurring 2 plasmid of yeast. These vectors contain DNA fragments from the 2μ yeast plasmid, yeast nuclear DNA, and the E. coli vector, pMB9. Other yeast vectors are recombinants consisting of E. coli vectors and yeast DNA fragments.

Bacteriophages

Bacteriophage lambda is a virus of E. coli. Several lambda derived vectors have been developed for cloning in E. coli, and for the isolation of particular genes from eukaryotic genomes. These lambda derivates have several advantages over plasmids: (1) thousands of recombinant phage plaques can easily be screened for a particular DNA sequence on a single petri dish by molecular hybridization. (2) Packaging of recombinant DNA in laboratory cultures provides a very efficient means f DNA uptake by the bacteria. (3) Thousands of independently packaged recombinant phages can be easily replicated and stored in a single solution as a "library" of genomic sequences.

Cosmids Plasmids have also been constructed that contain the phage cos DNA site, required for packaging into the phage particles, and ColE1 DNA segments, required for plasmid replication. These plasmids have been termed cosmids. After packaging laboratory cultures, the vector is used to infect a host. The recombinant cosmid DNA is injected and circularizes like phage DNA but replicates as a plasmid. Transformed cells are selected on the basis of a vector drug resistance marker. Cosmids provide an efficient means of cloning large pieces of foreign DNA and have therefore been used for constructing libraries of eukaryotic genomic fragments.

Animal cells In contrast to the wide variety of plasmid and phage vectors available for cloning in prokaryotic cells, relatively few vectors are available for introducing foreign genes into animal cells. In the latter case, the most commonly used vectors are derived from simian virus 40 (SV40).The SV40 genome consists of a small covalently closed circular DNA molecule for which the entire nucleotide sequence is known. Normal SV40 cannot be used as a vector, since there is a physical limit to the amount of DNA that can be packaged into the virus capsid, and the addition of foreign DNA would generate a DNA molecule too large to be packaged into a viral particle.

However, SV40 mutants lacking portions of the genome can be propagated in mixed infections in which a "helper" virus supplies the missing function. Propagating recombinants in eukaryotic cells using SV40 vectors imposes some limitations, such as the severe limitation on the size of gene segments that can be cloned in the small viral genome and the fact that only cells permissive for SV40 replication can be used. Bovine papilloma virus is another DNA virus that has been used as a vector in eukaryotic animal cells. The rat insulin gene inserted into the viral DNA has been used successfully to transform mouse cells.

Another system for the stable transfer of genes into mammalian cells involves cotransformation of cells with two physically separate sets of genes. For example, the introduction of the thymidine kinase (tk) gene from Herpes simplex virus to mouse tk cells results in stable and efficient transformation of cells that express the viral gene. In Drosophilia, transposable DNA elements, that is, DNA segments that are capable of moving from one position to another within the genome of a cell, have provided the basis for development of an efficient and controlled system of gene transfer.

DNA segments of interest can be transposed into germ-line chromosomes along with a transposable element to which the segment has been previously ligated.Plant cells Two systems for the delivery and integration of foreign genes into the plant genome are the Ti plasmid of the soil bacterium Agrobacterium and the DNA plant virion cauliflower mosaic virus. The Ti plasmid is a natural gene transfer vector carried by a tumefaciens, a pathogenic bacterium that causes crown gall tumor formation in dicotyledonous plants. A T-DNA segment present in the Ti plasmid becomes stably integrated into the plant cell genome during infection.

This property of the Ti plasmid has been exploited to show that DNA segments inserted in the T-DNA region can be cotransferred to plant DNA.Caulimoviruses belong to a group of plant DNA viruses that contain double-stranded DNA. Of these, cauliflower mosaic virus is the best studied for its cloning vehicle potential. The genomes of over 30 strains of cauliflower mosaic virus have been mapped by using restriction enzymes. Purified preparations of the cauliflower mosaic virus DNA can infect plant cells with relatively high efficiency. Cauliflower mosaic virus DNA is alos transformable after gene manipulation, which indicates that it might be possible to use cauliflower mosaic virus as a plant vector.

Cloned Gene Expression It is sometimes the aim of the genetic engineer to promote the expression of a cloned gene not only for the analysis of gene structure and function but alos for amplification of the synthesis of a desirable gene product. DNA cloning methods have enabled the genetic modification of bacteria and unicellular eukaryotes, rendering them capable of producing virtually any gene product of animal or plant cells.The aim of gene transfer experiments is to examine the mechanisms of gene regulation and expression in the context of normal cell differentiation. DNA sequences cloned by recombinant DNA technology have been transferred into fertilized mouse, Drosophila, and Xenopus oocytes by microinjection techniques.

In the mouse and Drosophila this system has allowed study of the expression and regulation of transferred gene sequences in specific tissues during normal cellular differentiation, and their transmission from one generation to the next. The Xenopus oocyte has been used mainly for analysis of transcription and translation of injected sequences.In the mouse, foreign DNA is injected into a pronucleus of a fertilized egg and subsequently becomes incorporated into the chromosomes of the diploid zygote. The injected eggs are then transferred to a foster-mother mouse, where normal embryonic development occurs.

Some of the progeny mice come to contain the foreign DNA in their cells, and the expression of the introduced DNA can be examined during development of these transgenic mice.A variety of recombinant DNAs have been injected into the mouse oocyte, including plasmids containing SV40 sequences, the Herpes simplex tk gene, a human alpha interferon cDNA, and beta-globin genes from a variety of animals.  The injected DNA has been shown to be integrated into the genomes of some of the transformants, and in many cases transmission of DNA from the original transgenic mouse to its progeny has also been demonstrated.

Applications Genetic engineering technology has made possible a wide variety of practical applications, several of which are discussed below.Isolation of specific genes Recombinant DNA technology has permitted the isolation and detailed structural analysis of a large number of prokaryotic and eukaryotic genes. This contribution is especially significant in the eukaryotes because of their large genomes. The methods outlined above provide a means of fractionating and isolating individual genes, since each clone contains a single sequence or a few DNA sequences from a very large genome.

Isolation of a particular sequence of interest has been facilitated by the ability to generate a large number of clones and to screen them with the appropriate "probe" (radioactively labeled RNA or DNA) molecules.Analysis of gene structure and function Genetic engineering techniques provide pure DNAs in amounts sufficient for mapping, sequencing, and direct structural analyses. Furthermore, gene structure-function relationships can be studied by reintroducing the cloned gene into a eukaryotic nucleus and assaying for transcriptional and translational activities. The DNA sequences can be altered by mutagenesis, before their reintroduction in order to define precise functional regions.Such analyses have provided insights into the complexity of the eukaryotic genome.

For example, many eukaryotic genes contain noncoding sequences (introns) interspersed in the coding sequences (exons) of genes. The primary RNA transcripts are spliced to remove the noncoding sequences are the mRNA matures. Exon-intron boundaries from diverse sources have been sequenced to reveal a common sequence pattern at the opposite ends of different introns. These sequences may be part of a mechanism that brings opposite ends of introns together prior to splicing.Cloning and sequencing of increasing numbers of eukaryotic genes have brought to light similarities in specific sequences near the 5' and 3' ends of different genes. These sequences are suspected to be involved in the regulation translation.

Molecular probes for a number of eukaryotic genes have revealed the existence of clustered gene families. Some families, such as the histones, have genes with nearly identical sequences which are arranged tandemly along the chromosomal DNA and which function simultaneous to synthesize their ,gene products. Members of other gene families, however, though clustered on the chromosome, are not identical; such genes encode closely related proteins. Embryonic and adult globin polypeptides are examples of such a gene family. Within the globin gene cluster are also found" globinlike" sequences that are nonfunctional because of deletions in essential regulatory regions.

These segments of DNA m y represent vestiges of globin genes that were once functional but, during evolution, have lost their physiological role. Polypeptide production Genetic engineering methodology ha provided means for the production of polypeptides and proteins. Although this expression of heterologous genes is function of a variety of complex factors, maximizing the expression of cloned sequences has been under intense and rapid development. It is now possible to produce a wide variety of foreign proteins in E. coli.

These range from enzymes useful in molecular biology to a vast range of polypeptide with potential human therapeutic applications, such as insulin, interferon, growth hormone, immunoglobins, and ezymes involved in the dynamics of blood coagulation. Biotechnology on a large scale ha evolved as a result of genetic engineering in the laboratory. Human insulin manufactured through the use of genetically engineered E. coli has been used safely in diabetes therapy. Other polypeptides such as human growth hormone an interferon have been in production. Research has been under way to manufacture vaccines and many other therapeutic and diagnostic agents by using the new technology.

Medical applications The screening of appropriately restricted human DNA with suitable molecular probes, that is , true genes or simply nonrepeated sequences of human DNA cloned in a vector, has disclosed differences between individuals with respect to the size of the restricted DNA fragment containing DNA sequences homologous to the probe. These types of variants within a population, called restriction-fragment-length polymorphisms, can be used to map the human genome.

Restriction-fragment-length polymorphisms are inherited by mendelian segregation and are distributed in populations asclassical examples of common genetic polymorphisms.

If such a DNA variant is located close to a close to a defective gene (which cannot be tested for directly), the DNA variant can be used as a marker to detect the presence of the diseases causing gene. It is estimated that detection of 150 to 300 different DNA markers of this type distributed randomly throughout the human genome would allow detection of any disease producing gene. Such information would be important in determining individual susceptibility to diseases and in taking to prevent them.

In cases where the specific mutation in DNA has been identified, such as sickle cell anemia, Certain restriction enzymes that recognize the abnormal DNA sequence at the mutant site can be used to demonstrate the mutation without study of the affected and unaffected family members. By using restriction enzymes in molecular hybridization experiments, changes in DNA sequences that occur in other hemoglobinopathies (genetically determine or synthesis of hemoglobins) may also be by using the appropriate probes. Such changes include deletions, mutations of restriction sites, and deletions of restriction sites. This kind of information antenatal diagnosis of human diseases by analysis of fetal blood samples.