Overview of Basic Tools and Methods for Genetic Engineering

Index

1. GENE CLONING PROCEDURES
1.1 RESTRICTION ENDONUCLEASES
1.2 ISOLATION OF DNA TO BE CLONED
1.3 C-DNA SYNTHESIS
1.4 GENE LIBRARY
1.5 RECENT TECHNIQUE
1.6 VARIOUS VECTORS USED IN r-DNA TECHNOLOGY
1.7 PLASMID CLONING VECTOR
1.8 CONSTRUCTION OF pBR
1.9 VIRAL DNA / BACTERIOPHAGES AS VECTOR
1.10 COSMIDS AS VECTOR
1.11 JOINING OF THE DNA WITH VECTOR
1.12 TRANSFORMATION AND GROWTH OF CELL
1.13 SELECTION OF CLONES
1.14 EXPRESSION OF CLONED DNA
1.15 PROTEIN PROTECTION

2. GENETIC MANIPULATION OF EUKARYOTIC CELLS
2.1. GENETIC MANIPULATION OF PLANT CELLS
2.2 GENETIC MANIPULATION IN MAMMALIAN CELL

3. BLOTTING TECHNIQUES
3.1. ANALYSIS OF DNA BY SOUTHERN BLOTTING
3.2 ANALYSIS OF PROTEIN BY WESIERN BLOT TECHNIQUES

4. DNA SEQUENCING
4.1 DNA SEQUENCING BY CHEMICAL DEGRACATION METHOD
4.2 SEQUENCING BY CHAIN TERMINATION
4.3 DIRECT DNA SEQUENCING BY USING PCR

1. GENE CLONING PROCEDURES

- The recombinant DNA technology involves the cloning of the higher organisms DNA into the prokaryotic organisms so as to allow these lower organisms to produce the specific product coded in the gene of higher organisms.
- The recombinant DNA technology is simply a “cut-paste mechanism” where a single gene is isolated from one organism (cut) and Inserted to another organism (paste) that will produce the new product or character (phenotype) according to the message (genotype) present in the gene.
- Among many enzymes, the most important are the enzymes used for cleaving the DNA, these enzymes are called Restriction Endonucleases (REN).
- These enzymes are the enzymes that have the capacity to break the DNA from phospho-diester bond between the two adjacent nucleotides.

1.1 RESTRICTION ENDONUCLEASES

- REN are the enzymes produced normally by various types of bacteria as a weapon against invading viruses. These enzymes can cleave the viral DNA and therefore they can restrict the further growth of viruses in that bacterium. Due to this character, they are called Restriction Endonucleases.
- Their existence was first identified by Werner Arber in early 1960s when he was studying on bacteriophages.
- Arber along with two other scientists Daniel Nathans and Hamilton Smith was awarded Nobel Prize in 1978 for their work with restriction enzymes.
- There are basically three types of REN found in nature…
- Type I REN
- Type II REN
- Type III REN
- Among these three types, Type II REN are most commonly used during gene cloning processes. Their cleavage is site specific.
- Their molecular weight is in the range of 20000-100000 Da. They contain two identical subunits.
- These enzymes can cut both the strands of DNA after recognizing specific nucleotide sequence.

illustration not visible in this excerpt

- Over 150 specific Endonucleases have been discovered which recognize more than 40 different sequences present on DNA.
- The specific palindromic identified by these enzymes are known as “Restriction sequence” which are palindromic in nature.
- The palindromic sequence when cleaved by REN, will generate two different types of the ends 1) Cohesive/Sticky / Staggered end & 2) blunt end.
- Cohesive ends have got certain, free single stranded nucleotides where as blunt ends do not have them.
- The nomenclature of REN is done according to the bacterial genera, species & strain in which the enzyme is produced. For e.g. ECoR1 is produced by the R1 strain of E.coli
- Various examples of REN are given above in the table.
- The 100% of specificity of REN to cut the DNA at only fixed sequence makes it very useful for using in R-DNA technology.
- The success of R-DNA technology is largely dependent on our ability to cut the DNA at specific defined sites.
- In this way REN play central & a key role in R-DNA technology.

1.2 ISOLATION OF DNA TO BE CLONED

- Generally microorganisms are used as the hosts for producing various biomolecules & metabolites of eukaryotic origin.
- Eukaryotic products are produced by the prokaryotic organisms when they have been given the proper DNA to produce the product.
- The DNA comes from a variety of sources.
- The isolation of DNA is very important first step of R DNA technology.
- One gene is a very small part of entire genome for e.g. one gene of E.coli = 0.03% of entire genome & one gene of mammalian genomes are approximately 0.03 x 10-3 % of entire genome.
- In this way to isolate the gene from a very large genome specifically in a correct manner is not an easy procedure.
- But the procedure becomes easy when m-RNA & not the DNA for the specific proteinic product is isolated.
- The procedure becomes easy because corresponding m-RNA is found when the protein is synthesized.
- The m-RNA can be isolated during

1)Hybrid release translation or
2)Hybrid arrest translation

1.3 C-DNA SYNTHESIS

The stepwise process of preparing c-DNA is explained below :

illustration not visible in this excerpt

- Reverse transcriptase enzyme has the capacity to catalyze the reaction of preparing DNA as per the sequences of RNA. The entire process will result into the synthesis of complementary DNA (c-DNA) from m-RNA. It is called c-DNA because it is not native DNA but it is produced from the spliced RNA.
- Eukaryotic genes contain non-coding intervening sequence known as “introns” which are removed from the hn RNA (heterogeneous nuclear RNA) after transcription and the coding sequences known as “exons” are joined together to form m-RNA. This process is called splicing of m-RNA.
- When we prepare c-DNA from spliced m-RNA, the c-DNA will not contain introns & so it will be different from native DNA which is a large DNA than c-DNA.
- This feature is very useful in prokaryotic gene cloning because prokaryotes do not have machinery for m-RNA splicing.
- However if one wants to study the native DNA, it would not be feasible to use c-DNA as it does not contain introns. Therefore except this purpose, c-DNA is widely used in R-DNA technology.

1.4 GENE LIBRARY

- In one crude method the entire DNA (genome) of mammalian origin is broken into pieces by multiple REN & all the pieces are cloned into microorganisms & ultimately by using the appropriate selection procedure, desired microorganisms are selected from many unwanted microorganisms. It is called preparation of Gene Library.
- This is a lengthy process because the selection of the desired microorganisms among many undesired microorganisms is time consuming & tedious process.
- Larger the genome & smaller the fragment size, the more colonies have to be grown.
- For e.g. The E.coli genome is broken down into 10kb pieces of DNA, total 1.5 x 103 pieces are generated. and if all are cloned in individual organism then 1.5 x 103 colonies are to be grown.
- If mammalian genome is broken into 10kb pieces of DNA, total 2 x 106 pieces are generated and therefore that many colonies are to be grown. This is a very large number and it is almost impossible to handle those and to select a single colony from that.
- Therefore, as a solution to this, the fragment size can be increased so as to decrease the no. of fragments.
- If the fragment size is increased from 10kg to 40 kb then the no. of fragments will be decreased & ultimately 7 x 104 colonies are grown and from them one will be selected
- The upper size limitation of vector to receive DNA is important to determine during the gene library because after DNA is fragment, the individual fragments are inserted in vectors & vectors will not receive the DNA having the size more than 40kg.
- The gene library is an older process but even in present day it is used for some specific gene cloning processes.

1.5 RECENT TECHNIQUE

- Nucleic acid chemistry has advanced today in such a manner it is possible to prepare the desired DNA molecules in the laboratory using modern technology and instruments.
- The process known as PCR technology (Polymerase Chain Reaction) is used to amplify the DNA and to generated DNA in laboratory.
- Even it is possible that we can obtain DNA if we have the proteinic product with us.
- Here we can sequence the protein and generate possible m-RNAs from that. Later on appropriate DNA can be produced from m-RNAs by reverse transcription.
- Such process had been successfully used for the synthesis of the hormone somatostain in bacterial host.
- In this way, the DNA can be isolated by various procedures and used further in recombinant DNA technology to allow the prokaryotes to produce the desired product.

1.6 VARIOUS VECTORS USED IN r-DNA TECHNOLOGY

- Vector is a molecule or organism which carries the DNA from one organism to another during R-DNA technology.
- It should allow the replication of the foreign DNA inserted in it, and moreover it should also allow the selection of an organism having vector by marking some product.
- These are ideal characteristics of a vector and additionally the vector should be able to carry the maximum possible length of the DNA.
- On the basis of these features, following vectors are useful during genetic engineering.

1)Plasmid as vector
2)Bacteriophages as vector
3)Cosmid as vector

1.7 PLASMID CLONING VECTOR

- Plasmids are small extra chromosomal circular DNA molecules which gie additional characteristic to the organism.
- Plasmids are separated from the main chromosomal DNA and they are separately replicated, independent of chromosomal replication (for this, they have their own origin of replication) and pass in the daughter cells during the cell division.
- Some very imp genes are carried by plasmids including antibiotic resistance, sexual factor, colicin, production, antibiotic production, toxin production, Nitrogen fixation, degradation of polymers, production of enzymes, cryptic gene etc.
- All these products are imparting additional features to the organisms that contain the plasmids.
- The presence of replication origins, markers gene and the frequent exchanging of genetic material between plasmids and chromosomal DNA make them very interesting for a biotechnologist who wants a vector during R-DNA Technology.
- Plasmids offer the convenient replication origin but there are many unwanted regions present on plasmids that can be removed when they are used as vector.
- The similar observations were made by two biotechnologists Bolivar and Rodriguez who prepared successful plasmid vector known as pBR-322 which is commonly used as vector during R-DNA Technology.
- Besides pBR-3zz (plasmid of Bolivar and Rodriguez), pBR-327 and PUC (plasmid of University of Callifornia) are also used in vector preparation.

1.8 CONSTRUCTION OF pBR322

- pBR-322 is a plasmid prepared in laboratory (in vitro) that contain the useful feature of different sources combined together to give a fully functional vectors pBR-322.
- As the requirement of certain characteristics is important in a vector, this plasmid has been prepared to fulfill all of them.
- It is the plasmid from which all unnecessary genes have been deleted & the gene having useful feature are only kept.
- Origin of replication has been taken from a plasmid occurring in E. coli known as Col E1.
- pBR-322 contains approximately 20 restriction sites and there are two antibiotic resistance genes in pBR-322 namely ampR and tetR (resistance against ampicilin and tetracycline respectively)
- Plasmid replication is not dependent on chromosomal DNA replication as plasmid is self replicating molecule having its own Ori C.
- It is possible to generate more copies of plasmid per bacterial cell by temporarily stopping the replication of main chromosomal DNA.
- Until the chromosomal DNA replication is completed, the cell will not divide but plasmid can replicate in this condition also, so copy no. can be increased.
- Practically copy no up to as much as 3000 plasmids can be generated.
- Due to the presence of marker genes the selection of plasmid is done easily.
- The presence of plasmid in bacterium will confer resistance to ampicillin and tetracycline.
- Furthermore, it is found that few restriction sites are present on the marker genes, so the plasmid DNA can be nicked from there to insert foreign DNA at this site and then the marker DNA is no ore functional.
- After recircularization of plasmid the two broken pieces of marker the gene will flank the foreign DNA.
- This will be the useful feature for differentiating recombinant plasmid (plasmids containing DNA in it) from other normal plasmids. This selection is illustrated in the next page in a diagram.
- The useful features of plasmid pBR 322 make it very useful for using as a vector during R-DNA technology.
- Smaller the size of plasmid, more are the chances of inserting large DNA into it.
- There are two reasons for using the smallest possible plasmid as a vector.
1.The small molecules of DNA are less easily damaged by shearing during isolation steps. This will allow the violent handling of plasmid during deproteinization and other steps of R-DNA Technology.
2.The chances of transformation increase as the size of plasmid decreases.
- Small molecules of plasmid are easily taken up by bacterial cells during transformation and large size will not allow it.
- Moreover, due to the smaller and smaller size of the plasmid, length of the foreign DNA can be increased to some extent.
- In this way pBR-322 is a very much useful plasmid cloning vector during R-DNA Technology providing all the necessary functions to a biologist.

1.9 VIRAL DNA / BACTERIOPHAGES AS VECTOR

- Although plasmids are excellent vectors but for larger pieces of foreign DNA, they have a size limitation.
- They cannot be much useful during the preparation of mammalian gene library. When the larger fragments of DNA must be cloned.

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