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The book deals with some of the important techniques of recombinant DNA technology including ; isolation of plasmids and transformation of bacteria, isolation and characterization of nucleic acids, construction and screening of gene libraries, DNA sequencing, RFLP and RAPD gene mapping, genetic transformation and molecular analysis of the transgenic plants.
The book includes protocols of some the important techniques currently in use in the field of plant biotechnology. The book also includes methods used in RFPL and RAPD analysis and a practical example of actual research problem in the field of plant genetic engineering. A glossary of important terms is also included which will be helpful to understand the subject matter. The preparation of common buffers and reagents used in the different protocols is included in the appendix.
0 Start Pages
Biotechnology is a broad discipline in which biological processes, organism cells or cellular components are exploited to develop value materials for the betterment of mankind.Technological advances influence the practice of science in a major way and available methods often dictate experimental approaches. Within the last three decades the developments of many powerful techniques and tools have resulted from the accumulated knowledge of molecular biology.
Biotechnology is a broad discipline in which biological processes, organism cells or cellular components are exploited to develop value materials for the betterment of mankind.Technological advances influence the practice of science in a major way and available methods often dictate experimental approaches. Within the last three decades the developments of many powerful techniques and tools have resulted from the accumulated knowledge of molecular biology.
1 Background - Plant Biotechnology
Plant biotechnology can be defined as the introduction of desirable traits into plants through plant tissue culture and genetic engineering techniques. The recombinant DNA technology is used to modify the expression of genes already present in the plants or to introduce new genes of other species with which the plant cannot be bred conventionally.
Plant biotechnology can be defined as the introduction of desirable traits into plants through plant tissue culture and genetic engineering techniques. The recombinant DNA technology is used to modify the expression of genes already present in the plants or to introduce new genes of other species with which the plant cannot be bred conventionally.
2 Isolation, Restriction and Agarose Gel Electrophoresis of E.coli Plasmid DNA
Plasmid is a double stranded, self replicating, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. The sizes of plasmids range from 1-1000 kbps. Plasmids are part of mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages and are associated with conjugation.
Plasmid is a double stranded, self replicating, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. The sizes of plasmids range from 1-1000 kbps. Plasmids are part of mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages and are associated with conjugation.
3 Transformation of Bacterial Cells
This is a procedure for transforming bacterial cells with circular DNA plasmids containing an origin of autonomous DNA replication and a selective marker (antibiotic resistance). Logarithmically growing cells are made permeable to DNA by incubation in CaCl2 and the DNA is taken up from the surrounding buffer.
This is a procedure for transforming bacterial cells with circular DNA plasmids containing an origin of autonomous DNA replication and a selective marker (antibiotic resistance). Logarithmically growing cells are made permeable to DNA by incubation in CaCl2 and the DNA is taken up from the surrounding buffer.
4 Preparation of Bacterophage ? DNA
Enterobacteria phage ? is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli (E.coli). It was discovered by Esther Lederberg in 1950. The wild type of this virus has a temperate life cycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.
Enterobacteria phage ? is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli (E.coli). It was discovered by Esther Lederberg in 1950. The wild type of this virus has a temperate life cycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.
5 Isolaton and Purification of Genomic DNA from Plants
There are various methods available for isolation of high molecular weight genomic DNA from plants. The basic steps involved in all these methods are removal of cell wall, nuclear membrane, proteins, lipids, carbohydrates, cell debris, RNA without affecting the integrity of DNA. The two major factors that affect DNA integrity are action of endogenous nucleases and impact of mechanical shearing.
There are various methods available for isolation of high molecular weight genomic DNA from plants. The basic steps involved in all these methods are removal of cell wall, nuclear membrane, proteins, lipids, carbohydrates, cell debris, RNA without affecting the integrity of DNA. The two major factors that affect DNA integrity are action of endogenous nucleases and impact of mechanical shearing.
6 Isolation of Total RNA and in vitro Translation of Poly (A)+ RNA
RNA is transcriptional product of DNA. The total RNA includes three classes, rRNA, tRNA and mRNA. The ribonucleic acids exists as ribonucleo protein particles in intact cells. The ribonucleo protein complex is dissociated by SDS in to RNA and protein deproutinised by phenol and the free RNA left in the aqueous solution is precipitated in cold by adding alcohol.
RNA is transcriptional product of DNA. The total RNA includes three classes, rRNA, tRNA and mRNA. The ribonucleic acids exists as ribonucleo protein particles in intact cells. The ribonucleo protein complex is dissociated by SDS in to RNA and protein deproutinised by phenol and the free RNA left in the aqueous solution is precipitated in cold by adding alcohol.
7 Isolation and Analysis of Polyribosomes and Ribosomal Proteins
Polysomes consist of two or more ribosome traversing a strand of mRNA, translating the nucleotide sequence into the corresponding amino acid sequence. Usually at any time, many ribosomes are translating an mRNA simultaneously forming a structure called a polyribosome or polysomes. One reason for isolating polyribosomes is to resolve them on sucrose gradients to determine the extent of ribosome loading. The second main reason is to provide a source of mRNA for in vitro translation.
Polysomes consist of two or more ribosome traversing a strand of mRNA, translating the nucleotide sequence into the corresponding amino acid sequence. Usually at any time, many ribosomes are translating an mRNA simultaneously forming a structure called a polyribosome or polysomes. One reason for isolating polyribosomes is to resolve them on sucrose gradients to determine the extent of ribosome loading. The second main reason is to provide a source of mRNA for in vitro translation.
8 Preparation of a Probe Radioactive Labeling of Nucleic Acids
DNA fragment to be used as a probe should be purified by gel electrophoresis. The band can be located after staining with ethidium bromide and electroeluted.
The in vitro radioactive labeling of DNA fragment is accomplished by the incorporation of a radioactive nucleotide during strand synthesis with Klenow enzyme (large subunit of DNA polymerase-I).
DNA fragment to be used as a probe should be purified by gel electrophoresis. The band can be located after staining with ethidium bromide and electroeluted.
The in vitro radioactive labeling of DNA fragment is accomplished by the incorporation of a radioactive nucleotide during strand synthesis with Klenow enzyme (large subunit of DNA polymerase-I).
9 Southern and Northern Blotting and Hybridization
Nucleic acid hybridization requires denaturation i.e. the breakage of H-bridges between the complementary strands of the DNA either by heating at high temperatures or by treatment with denaturing chemicals like formamide. The restricted DNA, therefore, is denatured before transfer from the gel onto synthetic membrane since only single stranded DNA binds to nitrocellulose.
Nucleic acid hybridization requires denaturation i.e. the breakage of H-bridges between the complementary strands of the DNA either by heating at high temperatures or by treatment with denaturing chemicals like formamide. The restricted DNA, therefore, is denatured before transfer from the gel onto synthetic membrane since only single stranded DNA binds to nitrocellulose.
10 Construction of Genomic Library
Exchange of genetic material rarely occurs between unreleated organisms and even similar species may not exchange chromosomal genes. A certain degree of DNA sequence homology is a prerequisite for genetic recombination in vivo. However, recombination between DNA molecules in vitro is not subject to such taxonomic restrictions.
Exchange of genetic material rarely occurs between unreleated organisms and even similar species may not exchange chromosomal genes. A certain degree of DNA sequence homology is a prerequisite for genetic recombination in vivo. However, recombination between DNA molecules in vitro is not subject to such taxonomic restrictions.
11 Screening of Genomic Library
Recombinant DNA is advancement in molecular technology which allows manipulation of genomes of higher organisms and dissection of their complex regulatory circuits. This new methodology is being used to understand the biochemistry of living organisms. The isolation of gene begins with the cloning of fragmented DNA of the organism into appropriate vector.
Recombinant DNA is advancement in molecular technology which allows manipulation of genomes of higher organisms and dissection of their complex regulatory circuits. This new methodology is being used to understand the biochemistry of living organisms. The isolation of gene begins with the cloning of fragmented DNA of the organism into appropriate vector.
12 Construction and Screening of cDNA Library
Complementary DNA (cDNA) is a double stranded DNA which is an exact copy of the mRNA, i.e. it consists of group of molecules possessing the information contained in various mRNAs but with termini equivalent to or related to those produced by restriction endonucleases. The use of cDNA is an absolute prerequisite for the expression of eukaryotic proteins in bacteria.
Complementary DNA (cDNA) is a double stranded DNA which is an exact copy of the mRNA, i.e. it consists of group of molecules possessing the information contained in various mRNAs but with termini equivalent to or related to those produced by restriction endonucleases. The use of cDNA is an absolute prerequisite for the expression of eukaryotic proteins in bacteria.
13 PCR and RT-PCR Methods
The polymerase chain reaction (PCR) is a very powerful new method used for in vitro to amplify a deoxynibonuclic specific region that lies between two regions of known DNA sequence single molecule to million folds in short time. This is achieved by an enzyme taq. DNA polymerase which is thermostable using this enzyme can synthesize microgram quantities of DNA of specified length and sequence from pictogram quantities of complex template in a few hours time.
The polymerase chain reaction (PCR) is a very powerful new method used for in vitro to amplify a deoxynibonuclic specific region that lies between two regions of known DNA sequence single molecule to million folds in short time. This is achieved by an enzyme taq. DNA polymerase which is thermostable using this enzyme can synthesize microgram quantities of DNA of specified length and sequence from pictogram quantities of complex template in a few hours time.
14 M13 Cloning for Di-deoxy DNA Sequencing
M13 is a single stranded filamentous DNA phage. Vectors have been developed from M13 phase by Joachim Messing (1983), which are suitable for preparation of single stranded DNA required for sequencing by Sanger’s method. These are derivatives of M13 wild type (size 6.407 kb).
M13 is a single stranded filamentous DNA phage. Vectors have been developed from M13 phase by Joachim Messing (1983), which are suitable for preparation of single stranded DNA required for sequencing by Sanger’s method. These are derivatives of M13 wild type (size 6.407 kb).
15 DNA Sequencing Methods
DNA can be sequenced in both double stranded and single stranded templates by cloning in plasmid or phage vectors. There are two sequencing methods.
DNA can be sequenced in both double stranded and single stranded templates by cloning in plasmid or phage vectors. There are two sequencing methods.
16 Isolation and in vitro Culture of Protoplasts
Protoplasts are cells which have had their cell wall removed, usually by digestion with enzymes. Cellulase enzymes digest the cellulose in plant cell walls while pectinase enzymes break down the pectin holding cells together. Once the cell wall has been removed the resulting protoplast is spherical in shape. Digestion is usually carried out after incubation in an osmoticum (a solution of higher concentration than the cell contents which causes the cells to plasmolyse). This makes the cell walls easier to digest. Debris is filtered and/or centrifuged out of the suspension and the protoplasts are then centrifuged to form a pellet. On resuspension the protoplasts can be cultured on media which induce cell division and differentiation.
Protoplasts are cells which have had their cell wall removed, usually by digestion with enzymes. Cellulase enzymes digest the cellulose in plant cell walls while pectinase enzymes break down the pectin holding cells together. Once the cell wall has been removed the resulting protoplast is spherical in shape. Digestion is usually carried out after incubation in an osmoticum (a solution of higher concentration than the cell contents which causes the cells to plasmolyse). This makes the cell walls easier to digest. Debris is filtered and/or centrifuged out of the suspension and the protoplasts are then centrifuged to form a pellet. On resuspension the protoplasts can be cultured on media which induce cell division and differentiation.
17 Methods of Genetic Transformation of Plants
Developments in recombinant DNA technology have made it possible to transfer gene from any source across species barrier. In fact, transfer and expression of foreign genes in plant cells is now a routine procedure. Of the several methods tried so far, the system based on Ti plasmid of Aprobacterium tumefaciens appears to be most successful for transformation of plant cells.
Developments in recombinant DNA technology have made it possible to transfer gene from any source across species barrier. In fact, transfer and expression of foreign genes in plant cells is now a routine procedure. Of the several methods tried so far, the system based on Ti plasmid of Aprobacterium tumefaciens appears to be most successful for transformation of plant cells.
18 Restriction Fragment Length Polymorphism (RFLP) and Random Amplified Polymorphic DNA (RAPD) Analysis
The genetic information which makes up the genes of higher plants is stored in DNA sequences of the nuclear chromosomes and the organelle genome. There is such an enormous amount of DNA in higher plant cell, that no two organisms are likely to be identical in DNA base sequence.
The genetic information which makes up the genes of higher plants is stored in DNA sequences of the nuclear chromosomes and the organelle genome. There is such an enormous amount of DNA in higher plant cell, that no two organisms are likely to be identical in DNA base sequence.
19 Development and Characterization of Transgenic Crop Plants
Example: Isolation and Characterization of Protease Inhibitor Gene and Promoter from Black gram (Vigna mungo L.) and Genetic Transformation and Molecular Analysis of Transgenic Tobacco
Example: Isolation and Characterization of Protease Inhibitor Gene and Promoter from Black gram (Vigna mungo L.) and Genetic Transformation and Molecular Analysis of Transgenic Tobacco
