In this article we will discuss about Modern Genetic Mapping:- 1. Introduction to Genetic Mapping 2. Methods of Genetic Mapping 3. Isolation of Complementary DNA (cDNA) 4. Recombinant DNA Technology 5. Cloning Vectors and their Nature 6. Shuttle Vector 7. Genomic Libraries.
- Introduction to Genetic Mapping
- Methods of Genetic Mapping
- Isolation of Complementary DNA (cDNA)
- Recombinant DNA Technology
- Cloning Vectors and their Nature
- Shuttle Vector
- Genomic Libraries
1. Introduction to Genetic Mapping:
Generally, Genetic Mapping is the method of using genetic crosses to locate genes on chromosomes relative to one another. In a conventional method, mapping is done using linkage groups. Genes located on the same chromosome are called linked genes or linkage groups.
The number of linkage groups in an organism is equal to the haploid number of chromosomes. With the help of test-crosses, the number of linked genes can be determined from which ultimately a linkage map or genetic map can be prepared.
This mapping is useful in the detailed study of any type of genetic analysis. This information can be useful to know the phenomenon of gene regulation. It can also help in Recombinant DNA research and DNA sequence studies. We want to discuss here some methods which are not related with linked genes.
2. Methods of Genetic Mapping:
(i) Mapping through Tetrad Analysis:
This method can be used to map genes in a few organisms where the product of meiosis is present in-a single structure. Tetrad analysis is possible in some fungi like Neurospora crassa, yeast and in algae like Chlamydomonas.
These organisms are haploid and, as there is no genetic dominance, the genotype is expressed directly in the phenotype. The usefulness of this method can be understood if we look at the life cycle of a few such organisms.
Life Cycle of Neurospora Crassa:
It is a fungus of the family Ascomycetaceae, producing a mycelial mat when it grows on the bread.
It is a classical material for genetical and biochemical studies for two important reasons:
i. It is a haploid organism, so the effects of any mutation can be detected.
ii. Life cycle is very short—so study of the segregation of genetic defects can be made.
A sexual reproduction of Neurospora is done through asexual spores, called condition. It can also reproduce by sexual method. There are also two mating types-A and ‘a’. A strains cannot mate with other A strains, similarly ‘a’ strains do not mate with other ‘a’ strains.
N. crassa reproduces sexually under normal conditions, but when there is a starvation of nitrogen sources in the medium, it reproduces through sexual means. Cells of the two mating types fuse to form a diploid nucleus (A/a type).
This diploid nucleus undergoes meiosis just after its formation and produces four haploid nuclei (2A and 2a) within an ascus. Then eight haploid nuclei are formed in the ascus through mitosis giving rise to 4A and 4a. So, the ascus contains the products of a single meiosis.
The unique feature in this type of fungus is that all the products of a meiosis are present in a sac in an orderly manner which can be isolated and cultured separately for analysis. Again, the order or arrangement of spores within an ascus can be correlated with the orientation of the 3 chromosomes of the first meiotic division (Fig. 17.1).
Yeast also shows the same type of life cycle like Neurospora. But the four ascospores are arranged randomly in yeast while is Neurospora these are linearly arranged. The data obtained from the culture of isolated spores (Tetrad Analysis) can be used for drawing genetic maps. Tetrad analysis is similar to that of test crosses in the conventional breeding methods.
A diploid zygote is first produced which is heterozygous for both genes and the tetrad analysis has been made after meiosis. Three types of tetrads will be formed if a cross is made between ab and ++. These tetrads are called parental type, tetra type and non-parental type.
The ascus of parental type contains two types of spores—ab and ++. The tetra type contains two parental types and two recombinant types. The non-parental type has two recombinant types only, without any parental type.
When the two genes are on different chromosomes, parental and non-parental types are produced without involving any cross-overs. If the two genes are not linked, the frequency of parental type tetrads and non-parental type tetrads will be equal.
The tetra type tetrads will be produced when there will occur a single cross-over between one of the genes and the centromere on that chromosome. The frequency of these tetra type tetrads will depend on the distance of the genes from the centromere.
When the two genes are present or linked on the same chromosome, parental type of ascus will form when there is no crossing over between the genes. A single cross-over between the genes will produce tetra type ascus having two parentals and two recombinant types. In case of double cross-over, various types of tetrads are produced depending on the number of chromatids involved in the crosses.
Two strand double cross-over produces parental type, three strand double cross-over gives rise to two parentals and two recombinants and four strand double cross-over produces 4 recombinants, i.e., non-parental type of tetrads (Fig. 17.2).
So, parental type of tetrads are produced when there is no cross-over or when there is two- strand double cross-over. Again non-parental types are produced when there is a four strand double cross-over. It can be stated that two genes are linked when the frequency of parental type is greater than non-parental types.
If we know the number of recombinants from the tetrad analysis, then the distance between the two genes can be measured by using the formula:
(ii) Gene Mapping in Human Chromosomes:
In case of human, it is not possible to do genetic mapping experiments like that of other organisms. For this reason, most of the genes in the human being have not been located on all chromosomes. The special technique of somatic cell hybridisation is being used to map both sex-linked and autosomal genes in humans. However, mapping of human sex-linked genes can be carried out by recombination analysis.
Genetic analysis of humans is sometimes done by examining the occurrence of characters in the family trees of individuals. This type of investigation is known as pedigree analysis. The critical study of the phenotypic records of the family will help to know the mechanism of inheritance of the gene (or genes) responsible for the phenotypic characters.
Prom these studies, geneticists can predict about the occurrence of some deleterious or fatal effect of the traits among children of the family.
This type of analysis is called genetic counseling. They can predict about the traits which occur due to single gene difference. But pedigree analysis does not show the location of genes on the chromosome.
Sometimes large number of pedigree analysis gives out a value for the frequency of recombination between the two genes involved and an estimate of genetic map distance can be obtained. Using this method some of the human genes like colour blindness and hemophilia have been mapped. One of the most suitable method in mapp human genes is by Somatic Cell Hybridisation Technique.
(iii) Somatic Cell Hybridisation Technique:
Somatic cells can be grown in vitro in presence of a suitable medium which can finally lead to the establishment of cell-culture line. But these cells have growth for a definite time. If the cell lines are derived from malignant tissues, then the cells will grow for indefinite period of time.
With the development of the technique of cell fusion, human cell is fused with the malignant cell of mouse to utilise these cells in the mapping of human chromosomes.
These hybrid cells can be utilised for Genetic Mapping of human chromosomes. In order to get uninterrupted cell divisions, cultured human cells, say fibroblast cells, are fused with an established tumour cell from mouse.
To obtain high frequency of cell fusion, some fusigenic chemicals like polyethylene glycol (PEG) are used. Once the fusion between cells occur, the fused cell may remain either as a heterokaryon or the two nuclei fuse to form a single nucleus with two sets of chromosomes.
The division of these fused cells give rise to clones of cells containing two sets of chromosomes of human and mouse. These hybrid cell lines are used to locate genes to particular human chromosomes. This is due to the fact that some of the human chromosomes are eliminated in the human-mouse hybrid cells.
Some stable hybrid cell lines are found with single human chromosomes where genes for a particular protein or some specific enzyme activities can be identified or mapped. One of the important criteria for this type of study is the selection of hybrid cells. The selection of hybrid cells is done- through complementation technique. This selection method can be explained using the HAT technique.
(iv) HAT Technique:
This technique is very useful as here hybrid cells can be easily selected which can grow in the HAT medium but the parental cells will not. The term HAT means Hypoxanthine-Aminopterin-Thymidine. These components are added in the animal medium.
(v) Principle of the Technique:
Cells synthesize their DNA from purine and pyrimidine triphosphates which are again produced from monophosphates of purine and pyrimidine. These are generally produced from the precursor molecules present in the cell.
There is an alternative way of synthesis of purines and pyrimidine’s through the enzymatic degradation of DNA and RNA. This is called salvage pathways. For example, cells cannot synthesise purines and pyrimidine’s from the precursors when the drug aminopterin is present. So the cells are totally dependent on the salvage pathway for the synthesis of purines and pyrimidine’s.
For selection of the hybrid cells in HAT technique, two genetically defective cells were taken (Fig. 17.3). The mouse cell line was taken with defects in the purine salvage pathway, i.e., lacking HGPRT– (Hypoxanthine Phosphoribosyl transferase) activity and human cell line with defects in pyrimidine salvage pathway, i.e., TK– (Thymidine Kinase activity).
But the human cell lines has HGPRT+ activity and mouse cell has TK+ activity. These two cell lines cannot grow in the medium containing Aminopterin as they cannot use the purine or pyrimidine pathway.
When the two cells are fused, hybrid cells will grow as the human chromosomes have HGPRT gene and the mouse chromosomes have TK gene. Hence the hybrid of cells will grow through complementation but parental cells will not.
Due to the presence of both HGPRT gene in mouse chromosomes and TK gene in human chromosomes in the hybrid cells, the salvage pathways of purine and pyrimidine synthesis will operate and normal DNA synthesis takes place. Thus, the hybrid cells can be easily selected from the parental cells using HAT technique (Fig. 17.3).
Reasons for Using Hybrid Cells in Locating Genes in Human Chromosomes:
i. Human and mouse chromosomes can be easily identified not only with the size but also with the characteristic banding patterns of chromosomes. Human chromosomes show variation in size while the mouse chromosomes are more or less uniform and small.
ii. Hybrid cells can be selected from the parental cells using HAT technique.
iii. Genetic markers can be used to locate genes with the help of some fluorescence labelled antibodies. Using some specific marker it can be shown that a particular marker is present when a specific chromosome is present.
Example for locating Gene in a Human Chromosome:
For example, there is a deletion of a part of one arm of the chromosome 6 in one cell line (cell line B). In another cell line there is no deletion (cell line A) [Fig. 17.4(a) and (b)]. The somatic hybrid of cell line A with mouse cell having intact chromosome 6 will show some enzyme activity or a new protein bands indicating that the genes present in chromosome 6 are responsible for that.
But the hybrid cells containing chromosome 6 with deletion of some of its part will show absence of some enzyme activity or some protein bands indicating that these genes are present on the deleted segments of chromosome 6.
With establishment of large number of cell lines of this type, a particular trait can be identified on particular human chromosome. In this way large number of genes have been identified on human chromosomes.
The location of genes as well as the polymorphic nature of the location of genes have been extensively studied with the use of sophisticated biochemical techniques for isolating and analysing the properties of nucleic acid molecules. Different techniques will be discussed here.
3. Isolation of Complementary DNA (cDNA):
Any work on genetic engineering or genetic manipulation is the isolation of the specific or desirable segment of DNA. If the objective is to isolate a gene, then it should be related with the gene product which is generally a protein. This protein can be confirmed by using antibodies specific to that protein.
It is also known that protein is synthesised from mRNA which is again derived from one strand of DNA. So, the ideal attempt will be to isolate the mRNA transcribed from a particular gene. If this mRNA codes for a major protein of the cell, then the cell extract will contain high levels of that mRNA.
New DNA can be synthesised from mRNA which is the reverse of transcription with the help of an enzyme called reverse transcriptase.
This enzyme is commonly found in RNA tumour viruses. Thus a single-stranded DNA (copy DNA or complementary DNA) is produced from a mRNA using a mixture of four deoxyribonucleoside triphosphates. Since mRNA has a poly-A tail of its 3′ end, a short oligo-dT molecule is to be used as primer for reverse transcriptase.
After synthesis of the first strand of cDNA, a poly-d-C tail is added to its 3′ end using the enzyme terminal transferase and dCTP. This enzyme will also put d-C tail at the 3′ end of mRNA. The original mRNA is then removed by alkaline hydrolysis, leaving the only single-stranded cDNA.
In eukaryotes the mRNA has the exon sequence only leaving the intron sequences after processing. Thus, cDNA will represent only exon sequences of the gene, but not the original complete DNA sequence of the gene.
This cDNA single-strand can be used for the synthesis of the second strand with the help of oligo dG primer and Klenow fragment of DNA polymerase I or T4 polymerase. But this reaction can also be catalysed by reverse transcriptase.
The final product will be double-stranded DNA, synthesised originally from mRNA. The best studied reverse transcriptase is AMV- Reverse transcriptase, which is isolated avian myeloblasts virus. Sometimes during the second strand synthesis of cDNA, hairpin loop structure is formed which is then open by using Si nuclease.
Important Conditions for cDNA Synthesis:
In case of first strand synthesis:
i. Correct composition of the enzyme storage buffer is essential.
ii. The final pH should be 8.3.
iii. Concentration of Mg++ ions are critical.
iv. Concentration of deoxynucleotide triphosphates should be greater than 50 nm.
In case of second strand synthesis:
i. Temperature of the reaction mixture should be kept at 15°C;
ii. Presence of 100 mM KC1 is essential to promote strand formation.
cDNA molecules thus synthesised can be inserted into plasmids to transform bacteria, which are then grown to give colonies, cDNA can also be used as a probe to search for making a gene library for the desired gene.
Once the cDNA has been synthesised, this can be used for making a clone for the continuous supply of DNA fragments, which is very useful for recombinant DNA technology. Nowadays Genes (DNA) are synthesised by different firms for mass-scale production for commercial use. This is known as gene machines or automatic DNA synthesizers.
(i) Gene Machine or Automatic DNA Synthesizers:
These contain some instruments which are automated to produce short sequences of single- stranded DNA (oligonucleotides). These machines axe manufactured by different companies like Vega Biochemicals, Bio Logicals etc.
But the method of manufacture follows a basic pattern. Some solid support systems like polymeric resins, silica gel, polystyrene etc. axe used. The silica gel is preferred because SiO2 has greater dimensional stability in organic solvents.
The first step is the formation of silane linkage between the SiOH radicals and an aliphatic silane, which is known as derivatising (Fig. 17.5). Following some sequential steps, nucleosides are bound to solid supports like resin beads which are then used as packing material in a small column. The amount is approximately 200 mg.
The gene synthesizing methods followed by the various manufacturers are generally common with some little variations in the use of values and in the number of solution containers. There are some changes in the sequences of reactions and type of reactants.
Electronic circuits and micro-processing controls are used to make the methods more precise. Nowadays, the complete sequence operation is put into a computer for improving its reliability. Another manufacturer, Bio-search, uses a high pressure column to perform synthesis of oligonucleotides.
(ii) Nucleic Acid Hybridisation:
Under the normal temperature and ionic conditions DNA remains in a duplex state’ (two- stranded structure) by the hydrogen bonds of the base pairs. The two-stranded structure can be separated (melted) by heating in a buffer solution or by increasing the pH of the solution. But if again the temperature is lowered or the pH of the solution is reduced the separated strands will join again or re-associate.
This fact was first shown by Julius Marmur and Paul Doty under suitable experimental conditions in 1960. This type of re-association of DNA strand is known as molecular hybridisation or nucleic acid hybridisation.
It may take place between the complementary strands of DNA or RNA or between the complementary strands of DNA and RNA. The technique of nucleic acid hybridizations has a great implications in the understanding of gene Structure and expression.
(a) Principle and Method of Nucleic Acid Hybridisation:
When DNA is kept in high temperature or when pH of the DNA solution is increased (alkaline pH), the hydrogen bonds of the A- T and G-C base pairs are disrupted and then the molecule separates into two strands. DNA molecules are then said to be denatured.
This denatured DNA also shows some’ important physical characteristics such as:
(i) Decrease in viscosity of the DNA solution;
(ii) Increase in density of the DNA solutions and
(iii) Showing more absorption of ultraviolet light at 260 nm region of the spectrum.
The study of the absorption characteristics of the DNA solution and its rate of dissociation can be easily made under the UV- spectrophotometer with a Temperature Controlling Unit. This additional unit of the spectrophotometer can gradually increase the temperature of the DNA solution which is present in the cuvette.
The relative absorbance of DNA at 260 nm with the increase in temperature of the sample can be monitored in the spectrophotometer (Fig. 17.6).
The increase in absorbance, known as hyper-chromatic effect, varies with the DNA samples of different organisms and even between species. It has also been noted that melting (denaturation) occurs over a small range of temperature like the melting of a crystal.
Hence the melting temperature or Tm is known as the temperature in which the denaturation of double-stranded to single-stranded DNA has done half-way to completion.
Of the base pairs present in DNA, G-C base pair is held together tightly by three hydrogen bonds, while A-T base pair is held together by two hydrogen bonds. Hence, the DNA sample with high G-C content will take more time to melt than DNA with low G-C content.
So, the first criterion in nucleic acid hybridisation is the dissociation of double- stranded DNA into its single-strand. This single-stranded DNA has the ability to form complementary hybrids with other single- stranded DNA or RNA molecules.
After denaturation of DNA or RNA, these single- strands are mixed together and incubated under suitable temperature in a suitable buffer solution. In addition to the nucleic acid hybridisation in solution, the hybridisation can be done on the surface of the membrane after immobilizing one single-strand of nucleic acid on it. Hybrid molecules can be visualised by various techniques.
(b) Hybridisation Under the Electron Microscope:
Single strand structure of nucleic acids can be differentiated from the double-stranded structure (duplex region) under the electron microscope. The duplex region of the DNA molecule is known as homologous region, which is differentiated from the non-complementary regions (single-stranded heteroduplex).
With the help of this technique, it is possible to locate DNA sites that are complementary to mRNA molecules, if single-stranded RNA is used to induce DNA-RNA hybridisation’.
The first step in the nucleic acid hybridisation is the dissociation of double-stranded DNA into single-strand (Fig. 17.7). These dissociated strands are then passed through nitrocellulose filter or nylon membrane where only single-strands of DNA will stick to the membrane.
In this way, one strand of nucleic acid is immobilised on the membrane surface. Another single-stranded DNA or RNA is at first labelled with some radioactive substances such as p32 and the membrane containing single- stranded DNA is incubated with radioactive DNA or RNA.
It has been noted that the complementary region with minimum 20 bases is sufficient to cause hybridisation of two nucleic acids.
Sometimes single-stranded region of one nucleic acid may be longer than the hybridized zone, hence the sample is treated with SI nuclease to degrade non-complementary single-stranded nucleic acids. Then the presence of radioactivity is demonstrated after exposure of the membrane to an X-ray film.
The technique of transfer of single-stranded or denatured DNA to the membrane was first developed by Edwin Southern and is known as Southern blotting. Similar techniques were developed for RNAs and proteins and were known as Northern and Western blots, respectively.
(c) Southern Blotting:
When isolated, pure DNA from various organisms is digested with restriction enzymes and electrophoresed in agarose gel. The gel showed a long smear of DNA fragments covering a whole spectrum of size in case of higher organisms.
The DNA is then denatured by treating with alkali and the single-stranded DNA was then transferred to strips of cellulose nitrate filter paper or Nylon membrane. In order to identify the particular fragment of DNA, it is necessary to the single-stranded and radioactively labelled DNA or RNA as probes.
The complementary DNA sequences will combine with probes to form a hybrid structure. After washing the membrane with buffer solution, the radioactivity can be demonstrated through autoradiography. Nowadays non-radioactive labelled probes are also used in Southern blotting technique where the specific fragments can be identified through fluorescence photography in the X-ray film (Fig. 17.8).
Conventional Southern Blotting technique uses radioisotopes p32 which have several disadvantages. Exposure to X-ray film require several days and probes must be prepared just before use due to radioactive decay. In addition to this, safety measures are difficult to maintain in all laboratories and disposal costs are continually increasing.
Non-radioactive labelling techniques for detecting nucleic acids utilizing alkaline phosphatase conjugates activates the chemiluminescent substrates (CSPD) producing light. The localised glow of light can be. imaged on X- ray film with film exposures of 5-60 minutes and subprogram quantities of DNA can be detected.
Oligonucleotide probes (biotinylated) can be directly conjugated with alkaline phosphatase. Indirect labelling procedures can be done with the use of biotin, fluorescein, DNP or digoxigenin into the nucleic acid probe.
Following incubation, the blot is incubated with streptovidin antibodies conjugated with alkaline phosphatase. This conjugate can now be used for chemiluminescent nucleic acid detection. This method has an advantage in having low background, hybridizations and rapid film exposures.
A single copy gene can be detected in 0.25 µg of human genomic DNA using an alkaline phosphatase labelled oligonucleotide. Similar procedure, known as Northern blotting, is utilised to analyse fractionated RNA samples in an agarose gel for detecting RNA molecules that are complementary to a radioactive nucleic acid probe.
(d) In Situ Hybridisation:
Hybridisation techniques can also be used to localize specific nucleic acid fragments that reside in their original site (in situ) within cells. This method is known as in situ hybridisation technique which has been applied by many research workers to locate DNA sequences coding for ribosomal RNA on chromosomes in cells or within tissue sections.
The majority of studies on in situ hybridisation were the use radio-iso-topically labelled probes which can be detected through autoradiography. Nowadays, the probe has been labelled with some fluorescent compounds using biotin which can be detected using fluorescence microscopy or immunological methods. This in situ hybridisation technology may be used to locate genes on chromosomes of an organism.
The technique of in situ hybridisation has also been used to identify the location of a specific nucleotide sequence in a population of cells. This method is known as colony hybridisation technique. In this method an agar plate containing bacterial cells is covered with a nitrocellulose membrane to take a blot of the pattern of colonies.
The membrane is then hybridized with a radioactive DNA or RNA probe and detected through autoradiography. This method is very simple and rapid to detect any specific activities using a probe. This technique has now been widely used in medical sciences as a quick diagnostic kit with the help of the recombinant DNA technology.
The synthesised DNA copies from mRNA through an enzyme Reverse Transcriptase is called complementary DNA or cDNA. The cloning of cDNA is a process of constructing DNA fragments from mRNA which is isolated from cells and these DNA copies are inserted into a cloning vector. Using this cDNA cloning, cDNA library can be made.
Usefulness of cDNA libraries:
The cDNA libraries are more useful than genomic libraries as the cDNA libraries are smaller than the latter. In other words, they consist of smaller number of clones, so they are easy to manipulate. The smaller size of cDNA library is due to the fact that eukaryotic genes have exons and introns.
Only exons are translated. cDNA library contains only exons, not introns. cDNA library can be generated from specific mRNAs at different developmental stages.
Procedures in Making cDNA Library:
Before making cDNAs, mRNAs are isolated from the cell and are then inserted into, the suitable cloning vector. The study and analysis of cDNAs are very useful as it reflects the expression of the gene (Fig. 17.20).
When RNAs are isolated from cells, these RNAs contain different types of RNAs like Ribosomal RNA, Transfer RNA, small nuclear RNA and messenger RNA (mRNA). Of all these RNAs, mRNAs can be characterised easily as it contains Poly A tail. These mRNAs are purified by passing the mixture of isolated RNAs through a column containing oligo (dT) chains, i.e., chains of de-oxy-thymidylic acid.
This type of column is known as oligo (dT) column. The poly-A chains of mRNA are attached with the oligo (dT) chains forming complementary base pairs. Thus mRNAs remain attached in the column, other RNAs will be washed away. Then these captured or bound mRNAs are released by increasing the ionic strength of the buffer.
(i) First method:
From these pure mRNAs, complementary DNA strands are prepared with the help of the enzyme Reverse transcriptase. The short oligo dT chain acts as a primer for the synthesis of DNA. But in this process a short hair-pin loop is produced at the 3′ end of a single- stranded DNA.
On alkaline hydrolysis, i.e., with the treatment of NaOH, the RNA strand is eliminated from the RNA-DNA hybrids. Now the 3′ end of the hair pin loop acts as a primer for DNA polymerase I for the synthesis of another DNA strand complementary to the first one.
Thus the double strand DNA molecule is produced which is complementary to the base sequences present in mRNA. The enzyme S1 nuclease is used to cleave the hair pin loop of the DNA molecule to produce an usual double- stranded cDNA molecule (Fig. 17.20).
It has been noted that some mRNAs are found in large quantities and others are found with few copies only. For example, typical mammalian cells have different mRNA molecules ranging from 10-30,000 types, depending on the number of copies present in the cell (Table 17.4).
Thus, all types of mRNA molecules are to be included in order to complete the cDNA library.
(ii) Second method (Okayama and Berg, 1982):
In this method, homo-polymeric tails are included in the vector for the insertion of cDNA. The vector generally used is pBR 322 which is opened with Pst I—thus eliminating the AmpR gene.
Then a short oligonucleotide containing guanine (i.e., oligo dG tails) (single-strand of DNA containing only guanine) is added to the 3′ end of the vector by using dGTP and terminal transferase (Fig. 17.21). Similarly, oligo dC tails are added to the cDNA molecule (3′ ends).
As these two types of tails in the vector and cDNA are complementary, the cDNA will be inserted into the vector easily to form a recombinant DNA molecule when the vector and cDNA are missed. But some gaps are present between cDNA and the vector as these are attached only between the oligo (dG) and oligo (dC) tails.
At this stage the vector is inserted into the host cell (E. coli) through transformation and the gaps are heated up by the E. coli enzyme. These new recombinant or trans-formant cells are tetracycline-resistant only as the cDNA is inserted in the site of the AmpR gene.
(iii) Third method:
In this case, linker is used during insertion of cDNA into the vector. The linker is a short double-stranded oligonucleotide, about 8-12 bp long, and has a restriction enzyme site. The isolated desirable cDNA molecule is mixed with the linker, say Bam HI linker, in presence of ligase. Bam HI linkers are added to each end of a cDNA molecule. But both the cDNA and the linkers contain blunt ends.
Then the cDNA-linker complex is cleaved with Bam HI restriction enzyme to produce this molecule with sticky ends (Fig. 17.22). The resulting cDNA-linker can be inserted into an appropriate cloning vector which is also cleaved with Bam HI. This recombinant DNA molecule is then inserted into E. coli cells through transformation for cloning.
Other Vectors Used for Cloning cDNA:
Nowadays, the common vectors used for the construction of large cDNA libraries are phage vectors such as λgt, 10 and λgt 11. These λgt 10 and λgt 11 and other series of λgt are derived from the λ vector. The map of the λgt is shown in Fig. 17.23 having cos ends at one end and there are six Eco RI sites designated from A to F.
Two Eco RI sites (at 39.7 and 45.6 Kbp site) of the wild genome are eliminated in λgt. After elimination, it was religated and recircularised to produce λgt — 0, λgt — AB’ and λgt — λc. These derivatives lack domains B and C, C or B respectively.
There is another deletion of nin 5 site from the wild type genome in λgt – 0. Hence, λgt — 0 lacks a total 29.3% of the wild genomic DNA. But this causes the DNA length of λgt — 0 to fall under lower DNA length necessary for phage encapsidation.
Hence at least 2.2 Kbp or at most 16.9 Kbp are to be inserted in this phage to get a progeny leading to a positive selection of recombinant phages after cloning and transformation into the host cells.
λgt 10 can take up DNA inserts up to 7.6 Kbp in length. The insertion of cDNA in CI gene gives rise to the formation of Cl-phages showing clear plaque in the culture plate. The λgt CI+ phages shows turbid plaques in the culture plate.
λgt 11 was constructed by Young and Davis (1983) and it contains lac Z fragment. About 7.2 Kbp DNA can be inserted in the Eco RI site. In the case of λgt 11, gene inserts, if any, can be selected by the presence of coloured plaques. When these are plated in the media containing mutant E. coli host (SupF lac 100), lac operon inducer IPTG and the substrate X-gal.
Another commonly used vector for constructing cDNA library is AZAPII which can take up 10 Kbp foreign DNA at 6 cloning sites and coloured plaques (blue) are selected for the recombinants. Recently, large quantities of cDNA molecules are generated with the help of Polymerase chain reaction (PCR).
Since the cDNA already contains polyd (T) tails at its 5′ terminus, amplification can be easily done by using oligo rd(T) and oligo d(C) as primers. Thus method is now very useful for marking cDNA libraries from a mRNA population from 1 or 2 mammalian cells.
Sometimes there are few in RNAs of low abundance, i.e., mRNAs present at less than 15 copies per cell. Consequently, complete cDNA library must include these mRNA molecules also. Generally mammalian cells contain 10- 40,000 different mRNA molecules. These different molecules of mRNA are present in different copies per cell (Table 17.5).
Very recently, large cDNA libraries are generated by using phage vectors such as λgt 10 and λgt 11 due to their efficiency and reproducibility of the in vitro encapsidation.
λgt 10 has a Eco RI site in the gene encoding the A repressor. This vector can accept fragments up to 7.6 Kbp in length. When the fragment is inserted into the vector, it inactivates the CI gene, resulting in the formation of clear plaques, while CI+ gene gives rise to turbid plaques. This property can be used for selecting the recombinant phages.
Another phage vector used is λgt 11 which has an unique Eco RI site at the terminal codon of lac Z gene. λgt 11 is also an expression vector. 7.2 Kbp of foreign DNA can be inserted into this vector at the position of lac Z gene.
It has also gene for temperature-sensitive repressor (cl857) which is inactivated at 42°C and an amber mutation (S100) occurs which prevents the phage from lysing host cells lacking the suppressor for this mutation.
The cloning methods (Fig. 17.24) in inserting cDNA into phage vectors, Agt 10 and Agt 11, are as follows:
i. Methylation of cDNA (ds) to prevent un- desired cleavage using Eco RI methylase.
ii. Ligation of synthetic adaptors (Eco RI adaptors in the figure) with the help of DNA polymerase I.
iii. Digestion with Eco RI to eliminate excess adaptors.
iv. Purification of modified DNA and its fractionation to get specific size (> 500 bp) by passing through O Gel A50m column.
v. Digestion of the phage vector with Eco RI and ligation to cDNA fragments.
vi. In vitro encapsidation.
vii. Cloning in E. coli.
When λgt 10 is used, recombinant vectors are selected from clear plaques. In case of λgt 11, recombinant ones are selected from coloured plaques when phages are plated on SupF lac I° host in presence of lac operon inducer IPTG and the substrate X-gal.
4. Recombinant DNA Technology:
We know that a typical eukaryotic cell contains at least a billion base pairs of DNA, while a single gene generally contains a few hundred to several thousand base pairs. So, the detailed study of any individual gene can only be made after the isolation of specific gene (DNA) from the whole genomic DNA.
The main methods for attaining this objective is through the use of Recombinant DNA technology which has come to the forefront as a result of the refinement of large number of techniques.
Actually, this recombinant DNA technology means the splicing of a DNA fragment from one organism and to insert this spliced DNA into another organism. This DNA is then known as recombinant DNA molecule. It is then cloned.
This recombinant DNA technology has a considerable application in biotechnology and genetic engineering for the production of antibiotics, hormones and other clinical applications in the diagnosis and the treatment of many genetic diseases.
W. Arber, H. Smith and D. Nathans first noted that bacterial cells produce enzymes that could cut up the double-stranded DNA molecules at specific locations. These enzymes can recognise a specific sequence and this sequence is different for enzymes isolated from different species and strains.
Such enzymes are known as restriction endonucleases or Restriction enzymes which can recognise specific DNA sequences 4-6 base pairs in length.
Restriction endonucleases were isolated during the study of the properties of certain viruses like bacteriophage λ. In the different strains of λ phage some restriction in the formation of plaque on E. coli cells (holes on a bacterial lawn) was noted.
These results were explained in noting the presence of some restriction endonucleases in that strain of λ phage. These enzymes have peculiar cutting properties. Their activity is dependent on the presence of Mg, ATP and S-adenosylmethionine.
These enzymes recognise specific sequences on double- stranded DNA, more 1,000-5,000 nucleotides away from the site and cut a single-strand DNA at a non-specific site liberating DNA fragments of about 75 bp in length.
These enzymes are known as Type I restriction endonucleases. As these types of enzymes could not cleave DNA at a specific site they are not used in Genetic engineering for the construction of recombinant DNA molecules.
Another type of restriction enzymes, known as Type II restriction endonucleases, was discovered in Haemophilus influenzae in 1970 (Smith and Wilcox, 1970; Kelly and Smith, 1970). These enzymes also recognise a specific nucleotide sequence in DNA and they break the DNA within that sequence.
In other words, these enzymes of particular Type II recognise particular sites on double-stranded DNA and cut always at the same specific sequence in DNA.
Hence, these are very useful for the construction of recombinant DNA molecules. Such enzymes are monomeric and require Mg++ for clearing the DNA. Large number of restriction enzymes of Type II have been isolated from different bacterial species. There are some enzymes which are inactive on host DNA but can cleave exogenous DNA molecules.
Rule of Nomenclatures for Restriction Enzymes:
The nomenclature for these enzymes was proposed by Smith and Nathans in 1973.
The following are the rules for their nomenclature:
a. The name was represented by three letters, of them the first capital letter represents the first letter of the generic name of the bacteria from which the enzyme was extracted. The second and third letters denote the first two letters of the species name.
They should be written in italics. For example, enzyme extracted from Escherichia coli is represented by Eco., Haemophilus influenzae by Hin., Haemophilus aegyptius by Hat.
b. Sometimes another letter is added after the third letter which represents the strain, bacterial type, virus etc. For example, Hind is Haemophilus influenzae Rd, EcoR is Escherichia coli Ryl3 etc.
c. If the strain from which enzymes are isolated has different restriction-modification systems, they are represented in Roman numerals such as Hind I, Hind III, etc.
Some properties of the two classes of restriction enzymes are given in Table 17.1.:
Properties of the Recognition Sites:
The recognition sites of Type II restriction enzymes are different and specific. Most of the enzymes recognise a sequence of 4, 6, and sometimes 5 nucleotides. These sites show palindromic sequences. That means the nucleotide sequence of any strand is identical to that of the complementary strand when they read along 5′-3′ direction.
For example, in case of Bam HI, the recognition sequence is GGATCC in the 5′-3′ direction which is identical in the complementary strand:
Type II enzymes have an axis of symmetry through the mid-point of the specific sequence, so the base sequence of one DNA strand in 5′-3′ direction is the same as the base sequence from 5′-3′ direction in the complementary strand.
Cleavage of a DNA fragment shows three types of configurations:
1. Some enzymes like Hae III or Smal cut DNA strands in the middle of the recognition sites in such a way that it gives rise to blunt ends:
2. Some enzymes like Bam HI, Eco RI etc. produce staggered or sticky ends or cohesive ends as shown in Fig. 17.9.:
3. Some enzymes like Pst I produces overhanging sticky ends of 3′ hydroxy extension.
Restriction fragments with sticky ends are of great value in cloning DNA fragments in the vector since these restriction enzymes make symmetrical cuts in DNA with sticky ends. DNA fragment produced by Eco RI or Bam HI with sticky ends can join easily with other DNA fragment produced by the same restriction enzymes like Eco RI or Bam HI with the help of ligase.
Characteristics of some restriction enzymes are given in Table 17.2.:
Restriction enzymes are generally stored at -20°C in 50% glycerol buffers. These enzymes are not stored in frost-free freezers, where cycle of heating goes on to remove the ice build-up. The unit of the restriction enzymes is defined as the amount of enzyme required to digest 1 µg of A DNA in 1 hour at the specified temperature and buffer conditions.
The restriction enzyme activity is measured through λ digestion test. DNA, after digesting with the restriction enzymes to be tested, is electrophoresed on agarose gels. For example, Eco RI digested λ DNA shows 6 fragments on an agarose gel after staining with ethidium bromide.
These fragments show molecular weights of 21.8, 7.5, 5.8, 5.5, 4.8 and 3.4 Kbp. The efficiency of digestion depends on the structure of DNA to be cleared. The supercoiled conformation of pBR322 plasmid DNA requires 5-10 Eco RI units for its complete digestion.
Conditions of Reactions of R.E.:
The digestion through restriction enzymes is generally carried out at 37° C with the exception of a few enzymes, such as Bst EII at 60°C. The reaction media consist of Tris-HCl buffer at neutral pH containing some salts like MgCl2, KCl and NaCl. The pH and ionic strength of the buffer vary from enzyme to enzyme.
Some enzymes work better at low salt concentrations (0-50 mM NaCl) while others work better at high salt concentrations (150mM NaCl). DNA digestions are stopped by the reaction buffer at 65-70°C for 5 minutes or by adding metal ion chelator EDTA.
The modified activity of certain enzymes, known as star activity, is found in case of certain enzymes when the conditions of the reaction buffer is modified, i.e., by using very high enzymatic concentration, using Mn++ ions instead of Mg++ ions, high pH low ionic strength and high concentration of glycerol.
Under this modified condition, the recognition sequence of the enzyme is modified. For example, the star activity of Eco RI* will make the altered recognition sequences such as AATT instead of GAATTC for the normal type of Eco RI.
5. Cloning Vectors and their Nature:
(a) Phage vector of A-vector:
Phage or Bacteriophage (or even A) is an E. coli phage. It is used as one of the first vectors in cloning purposes as the molecular genetics of this phage, such as its replication, gene regulation etc. have been studied in detail. It is a double-stranded DNA virus with about 50 genes.
One characteristic feature of this DNA is the presence of two short complementary sequences at two ends. These sequences consist of 12 nucleotides (5′-GGGCGGGCGACT-3′) which is known as coscohesive end.
It is a temperature phage having two different life cycles: lytic and lysogenic. In the lytic cycle, the λ chromosome (DNA) starts replication after entering the host cell (bacteria). The breakdown of host DNA takes place. The production of progeny phage or pro-phage starts with the lysis of the bacterial cell.
In case of lysogenic cycle, the phage DNA integrates into the host chromosome and then the replication of that DNA and bacterial multiplication goes on. Lastly, the exclusion of λ DNA (chromosome) takes place and then the lysis of the bacteria starts (Fig. 17.10).
Recent regulations of Genetic Engineering technology recommend using cloning vectors that does not follow the lysogenic cycle. Hence the specially engineered λ vectors have been made that can follow only the lytic pathway.
Secondly, these λ cloning vectors must have some restriction enzyme sites so that specific DNA fragments can be cloned in the vector. One such constructed λ vector has a left arm and right arm that contain all the genes needed for the lytic cycle.
Between the two segments there are some cleavage sites for one restriction enzyme by Eco RI. There should not be any other Eco KI site in that DNA. This site of DNA is called the replacement vector’s DNA.
Normally wild type A DNA contains 5 Eco RI sites. Several mutants are to be developed to get a λ cloning vector with 1 Eco RI site, or with 2 or 3 sites of different restriction enzymes through detection of Eco RI fragment.
Detailed genetic and physical map of λ DNA shows that wild type λ carries sites for the restriction enzymes Xbal and Xhol in the nonessential region of the genome. For the cloning of small DNA fragments, these fragments may be inserted at a unique restriction site located in the non-essential region of the genome. There are also Sail and Sst I sites in a non-essential region of the genome.
(ii) Construction of λ-Derivatives:
About 100 A derivatives have been made. The method of constructing the derivatives for inserting large DNA fragments is described below. In this method, the most commonly used derivatives of λ gt cloning vector have been made.
The linear λ DNA in Fig. 17.10 shows the location of 6 Eco RI sites and one cos site at one end. These 6 Eco RI sites can be designated as A to F. The deletion of certain restriction sites and recircularisation of Agt DNA results in the construction of vectors Agt-0, λgt-XB, λgt- λ c. These 3 derivatives have no B and C segments, C and B, respectively.
Another important derivative of λ vector is the Charon vectors. Charon 16A contains amber mutations in genes A and B and a substituted lac 5 fragment from the β-galactosidase lac Z gene that bears Eco RI and Sst I sites (Fig. 17.11).
The characteristic feature of Charon vectors helps in the colour selection of recombinant phages. The activity of the lac 5 gene can be directed by adding lac operon inducers IPFG or IPTG to the growth medium.
The effective enzyme can hydrolyse the substrate 5-bromo-4-chloro-3-indolyle- β-D- galactoside (X-gal) which produces a blue pigment. It is used as a marker. When any foreign DNA is inserted at the Eco RI or Sst I site, no active enzyme is produced, X-gal is not hydrolysed and so no colour develops.
In other words, when Charon 16A is cleaved with Eco RI or Sst I enzyme and is allowed to infect bacteria cells plated on the medium with IPTG and X- gal, it will develop blue colour (blue plaques) if there is no incorporation of foreign DNA.
But if there is an insertion of foreign DNA, colourless plaques will be found—indicating the presence of recombinant vector. Charon 16A is used when the recipient strain carries a lac mutation.
Another Charon vector, i.e., Charon 30, is one of the most versatile λ derivatives. Of the latest constructed λ vectors, Charon 34 and 35 are most sophisticated. These vectors can insert foreign DNA between 9 and 20 Kbp and the recombinant phages can grow on rec– strains.
The total length of λ DNA is of about 45 Kbp that can be packaged into λ DNA particles. The replacement vector’s DNA can only accept DNA fragment of 15 Kbp between the left arm and right arm of λ DNA (Fig. 17.12).
Plasmids are circular, small double-stranded DNA that replicate autonomously within bacterial cells. Most of the plasmid DNA can code certain specific characteristics of the cell, such as antibiotic resistance, toxin production, resistance to heavy metals, degradation of complex organic compounds etc. But there are certain plasmids which do not encode any phenotypic characters.
These are called cryptic plasmids. Plasmids vary from 2-200 Kbp in size. F plasmid of E. coli has the property of transferring genes from one cell to the other in having a set of genes, known as tra. Every plasmid has a constant copy number in each cell, but it can vary greatly. In case of gram-negative bacteria, two groups have been noted like—copy number and high copy number.
(a) Low Copy Number Plasmids:
Plasmids that have 1-5 copies per chromosome are called low copy plasmids, example -pSC 101.
(b) High Copy Number Plasmids:
Plasmids are those existing more than 15 copies per chromosome, example pBR322.
An inverse relationship has been found between plasmid copy number and plasmid size.
Naturally occurring plasmids are those plasmids whose construction was not made for cloning purposes. The plasmids used for cloning experiments are all derivatives of naturally occurring plasmids.
(c) Characteristics of the Plasmid Cloning Vector:
i. Presence of an Ori sequence which controls the replication of the plasmid within bacteria (E. coli).
Ii. Easily selectable phenotype or marker.
The common selectable marker is the presence of antibiotic resistance characters Table 17.3. For example, if a plasmid has a resistance character to two different antibiotics (ampR genes for ampicillin resistance and tetR genes for tetracycline resistance), the selection of recombinant plasmids can be easily done.
When a foreign DNA of desirable characters is inserted into a unique restriction site located in the gene encoding antibiotic resistance, say ampR, and is then injected into the host cell (bacteria), the plasmids containing gene insert or the transformed cells will grow in the presence of tetracycline through replica plating technique. The same transformed cells or colonies will not grow in the media containing ampicillin.
iii. Presence of restriction enzyme sites— Table 17.3 specially in the genes encoding selectable phenotypic markers will be the site for the insertion of new DNA fragments that are to be cloned (Fig. 17.13).
Plasmid pBR322 is the ideal plasmid which possesses all the characteristics and has been widely used as a cloning vector.
iv. Replication and amplification capabilities in the host.
v. Non-conjugativity (no transfer of DNA between individual cells).
(d) Plasmid pBR322:
This plasmid is 4363 nucleotide pair long, i.e., 4.363 Kbp. It has two antibiotic resistance characters, ampR and tetR genes and has several restriction enzyme sites like Eco RI, Hind III, Bam HI, Sal I, Pst I, Pvu I, Pvu II, Ava I and Clo I. These are the places where foreign genes or DNA fragments are inserted for cloning (Fig. 17.14).
TetR gene location in the vector contains sites for Hind III, Bam HI and Sal I and ampR gene location contains sites for Pst I and Pvu I. For example, if DNA fragments are inserted into the Hind III or Bam HI region, it will inactivate the tetracyclic resistance gene. Other restriction enzyme sites are not used for cloning experiments as they will be difficult to select using antibiotic resistance markers.
DNA inserts of 5-10 Kbp long are easily cloned in pBR322. Larger DNA fragments cannot be inserted in the pBR322 plasmids as the plasmids are not able to maintain the replication rate and stability due to increase in their size (Fig. 17.15).
Plasmid pBR322 was constructed through different steps from plasmid pMBl, pS 101 and pRSF 2124. Thus pBR 322 was constructed with the combination of some characters from different plasmids.
The characteristics are:
i. Origin of replication of pMBl.
ii. Ampicillin-resistance gene from pRSF 2124.
iii. Tetracycline resistance gene from pSC 101.
(e) Other Plasmids:
In some cases cloned gene products may be lethal to the host cell when plasmids are present in large numbers in bacteria. It is, therefore, useful to clone genes in low copy number plasmids like pSC 101. But pSC 101 carried only one selectable marker.
Thus several derivatives of pSC 101 were constructed for cloning such special type of gene inserts (Fig. 17.16). For example, pHSG 415 has three antibiotic- resistance genes (streptomycin, kanamycin and chloramphenicol) and has 5 copies per cell.
(f) pBR 322 Derivatives:
With the deletion of Hae II fragments from pBR 322, a new vector pAT 153 was constructed having threefold increase in copy number. Again the removal of nic site, which was present in Hae II fragment, leads to the loss of characteristics of mobilisation of the plasmid.
This has a great advantage because the new plasmid pAT 153 would not be transmitted to other bacterial strains. For the inclusion of the Eco RI site in pBR 322, Bolivar and co-workers constructed a series of plasmid suitable for Eco RI cloning (e.g., pBR 324, pBR 325, pBR 328).
(g) pUC Plasmid Series:
Taking the Pvu II-Eco RI fragment of pBR 322, pUC plasmids were constructed with Ampicillin-resistance gene, Hae II site and origin of replication. The multiple cloning site or poly-linker isolated from the M13 mp phage was inserted into the lac region of the plasmid.
The pUC plasmids within the lac- strains of E. coli show blue colonies. But when foreign genes are inserted within the multiple cloning site of pUC plasmids, they show white colonies due to the inactivation of lac genes. The common pUC plasmids used in the cloning experiments are pUC 8, pUC 9, pUC 18 and pUC 19.
A small cloning vector (4 Kbp) was constructed in the pEMBL series for the stable insertion of large DNA fragments. These pEMB2 vectors contain part of the lac Z gene and part of the β-lactamase gene with multiple cloning sites. These plasmids have the two origins of replication—one normal Col El origin, and the other f1 origin of replication from phage. Such plasmids with two .origins of replication are known as phagemids.
Cosmids are small plasmids (5 Kbp in size) with λ cos site. Its name signifies the hybrid nature of plasmids and bacteriophages showing the characteristics of plasmids and bacteriophages. The presence of cos site gives the property of bacteriophage encapsidation in vitro.
Long DNA fragments, ranging from 30-47 Kbp, can be cloned in cosmids while plasmid and λ cloning vectors can accept DNA fragments of 5- 10 Kbp and 15 Kbp, respectively. Cosmids are fully constructed in the laboratory by combining the characteristics of pBR 322 and phage λ.
Characteristics of Cosmid cloning vectors, such as cosmid pJB 8 (Fig. 17.17) are:
i. Size small, such as 5.4 Kbp.
ii. Has cos site.
iii. Origin of Replication is Ori.
iv. Presence of Ampicillin-resistance gene (ampR).
v. DNA fragments can be cloned in the Bam HI site.
As the first constructed cosmid had a small cloning site with one selective marker, several modifications of cosmids were made. For example, cosmid Mu-A-3 was constructed to have a cos site in a 403 bp Hinc II λ fragment cloned in the Pst I site of pBR 322.
Again, cosmid Homer I was constructed from the Charon 4A and pBR 322. Cosmids Homer V and VI contained Hpa II, Hind III fragment with SV4o origin of replication. Another cosmid pGcos 4 was constructed with a resistance characteristic to methotrexate due to the presence of dihydrofolate reductase (DHFR).
Cosmids are used for the cloning of large DNA segments—35-40 Kbp in length—and are very helpful for the construction of genomic libraries. When a foreign DNA of 40 Kbp is inserted into the cosmid vector, the recombinant cosmid is of the right size to be packaged into phage head (Fig. 17.18).
Thus the cosmids are advantageous because they are very useful to clone the large fragments of DNA. These vectors are very useful for the construction of several gene libraries like Saccharomyces, Drosophila and Mouse.
The cloning efficiency of cosmids is 103-106 transduced particles per fig of DNA which is higher than plasmids. For the stability of the cosmids, rec A hosts are used. One disadvantage in using cosmids for the construction of genomic libraries is the fact that sometimes there is a loss of cosmids having large DNA fragments.,
6. Shuttle Vector:
It is one type of cloning vector that contains selected markers for its detection in one or two host organisms. For example, YEp 24 is a shuttle vector which can be introduced both in yeast and E. coli.
Characteristic features of YEp 24:
i. Presence of Ori sequence so that it can replicate in E. coli.
ii. Presence of Tetracycline-resistance gene (TetR) to use as selectable marker in E. coli cells.
iii. Presence of URA 3 gene to act as a selectable marker in yeast.
iv. Presence of Autonomous replication sequence, so that it can replicate in yeast.
7. Genomic Libraries:
When the cloning vectors and restriction enzymes axe available, scientists tried to clone individual genes of an organism. The first step in this process is to isolate high-molecular weight DNA from cells or tissues of an organism and to fragment that DNA in different sizes by using restriction enzymes. These DNA fragments are then inserted into the suitable cloning vectors and cloned.
The Genomic Library is the collection of randomly cloning genomic DNA of a given organism into bacteriophage vectors (A) that includes all of the DNA sequences of a given species. In generating a genomic library, there should be no specific discrimination against a particular sequence.
When the library contains at least one copy of every possible sequence of the genome of an organism it is known as complete. The size of a library depends on the haploid content of DNA in the organism. Human and mouse genomes of 3 x 109 bp length require at least 2 x 105 bacteriophage vectors to include all the fragments 2 x 104 bp long.
Method of Producing Genomic Libraries:
(i) Earlier Method:
Genomic DNA of an organism is completely digested by the restriction enzyme and these fragments of DNA are incorporated into suitable cloning vectors and cloned in the host cell.
But in this method genes were split into number of small pieces which require a large number of cloning vectors for DNA of eukaryotic organism. The screening of specific genes was also very difficult as the genes were split into large number of pieces.
(ii) Recent Method:
Most of these problems are avoided by cloning larger DNA fragments (18-22 Kbp) in Cosmids. High molecular weight DNA isolated from an organism is sheared to fragments through mechanical stress (passing through a hypodermic syringe and needle).
The next step is the isolation of fragments that are neither too big or too small (18-22 Kbp). The isolation of these fragments is done either through sucrose density centrifugation or by electrophoresis through agarose gels.
Cloning is then performed into a vector of λ derivative which can insert the passenger DNA molecules up to 22 Kbp long. After digesting the vector with suitable restriction enzyme, the fragments of genomic DNA is ligated with the liberalized vector. The ligation is done with high genomic DNA concentration in order to get efficient ligation reactions.
For example, DNA is digested with the restriction enzyme Sau 3A which has the recognition sequence of 5′-GATC-3′. These ends are complementary to ends of the DNA of the cloning vector if it is digested with Bam HI, which has a recognition sequence of 5′-GGATCC-3′.
In the ligating solutions, the cut fragments of Sau 3A will join with the cut fragments of Bam HI of the vector DNA. The recombinant molecules thus produced can be cloned into the host cell (bacterium) (Fig. 17.19).
The colony containing gene insert or the gene library is preserved by growing the selected colony into the medium at low temperature. But, it is very difficult to represent all the sequences of the genome of an organism in the gene library which actually depends on the location of the restriction site in DNA.
If the restriction sites are very close or far apart, the chance of obtaining suitable size of DNA for cloning into the vector may be small. There may even be deletion of some sequence during processing.
Hence, the probability of DNA sequence to be included in the genomic library may be calculated from the formula:
For example, in a library of 40 Kbp fragments of the total genomic DNA 14,000 Kbp, the amount of recombinant DNA molecules would be required for 99% chance may be calculated as:
That means 1,610 recombinants of 40 Kbp size are needed to make a genomic library of 14,000 Kbp DNA.
Characteristics of an Ideal Genomic Library:
An ideal genomic library must contain all the DNA sequences of the organism. Each cloned fragment must not be too large or too small and is capable of mapping again through restriction enzymes. These fragments must also contain some overlapping regions for chromosome walking. It must have the capacity to overcome the storage conditions.
Once the new or desired gene is inserted in the cloning vectors, large number of these genes are obtained by culturing the vector in the host cell (bacterium). Recombinant DNA molecules have been obtained in plant, animal and human systems for further biochemical studies. These recombinant DNA technologies can also be used to make proteins that are necessary in agriculture, medicine and for other purposes.