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Introduction to Genetic Engineering What is Recombination and Recombinant DNA?
The resultant DNA formed as a consequence of collision of two or more DNA sequences is known as recombinant DNA. The process of joining DNA fragments by means of formation of phosphodiester bonds between them is called as recombination.In eukaryotes, recombination occurs during meiosis at pachytene stage where crossing over occurs between homologous chromosomes.
In prokaryotes recombination occurs through 3 different modes of gene transfer
The resultant DNA formed as a consequence of collision of two or more DNA sequences is known as recombinant DNA. The process of joining DNA fragments by means of formation of phosphodiester bonds between them is called as recombination.In eukaryotes, recombination occurs during meiosis at pachytene stage where crossing over occurs between homologous chromosomes.
In prokaryotes recombination occurs through 3 different modes of gene transfer
- Conjunction
- Transformation
- Transduction
Conjunction: In conjunction there is transfer of F-plasmid from a donor bacterium to a recipient bacterium through cell to cell contact.The transfer of DNA from F+ cell to F- cell is in single stranded form and this transfer of DNA is mediated by proteins coded by tra region of F plasmid. F plasmids has dual existence, they can either exist independently as F+ plasmids or get integrated into the chromosome (Hfr). Insertion of episome into chromosome is called as integration, which is a precise process and mediated by an enzyme called integrase. Reverse of this process is mediated by another enzyme called as excisase. Excission is not a perfect process because during this process apart from plasmid DNA adjacent chromosomal DNA regions are also excised. When these plasmids carrying the chromosomal DNA along with plasmid DNA integrates into another recipient bacterium results in recombination of those regions. The F- cells are called as High frequency recombinant (Hfr)
Transduction is a process of DNA transfer from one bacterium to the other through the virus leading to recombination. Viruses that infect bacteria are called Bacteriophages. Bacreriophages adsorb onto the membranes of the bacteria, drill a pore into the cell membrane through which they introduce the DNA into the cytoplasm of the host bacteria, which gets circularized immediately and subsequently integrates into the host genome and remains dormant. This is called Prophage which can get replicated along with the chromosomal DNA for generations Upon sensing the favorable conditions, the DNA excises itself from the chromosomal DNA, replicates by rolling circle mode of DNA replicate to produce multiple DNA molecules which gets packaged into viral particles and are released by lysing the bacterial cell. These progeny bacteria when infect other bacteria, same procedure is repeated, but this time the introduces viral DNA has certain chromosomal regions from the previous bacterial host which undergo recombination with the chromosomal regions of the new host.
Transformation:
Transformation is a process of introducing exogenous DNA into cells without the aid of any biological agent. Introduction of DNA leads to the change in the genotype which further results in the change in the phenotype. The ability of an organism to respond to externals signals is called Competence. Some species of bacteria like Streptomyces species are naturally competent and undergo transformation naturally whereas others are made competent by physical or chemical methods. Physical methods include electroporation which utilizes electric current in short pulses to introduce the DNA. Chemicals like salts of divalent cations such as Calcium chloride, Lithium chloride etc are used to make the cells competent. Such cells when given a heat shock tend to allow DNA molecules into the cells.
Transformation is the most frequently employed method in molecular biology to introduce the plasmid vectors into the bacterial cells. Plasmid vectors carry genes encoding antibiotic resistance which can be used as selection markers to select the cells that have undergone transformation.
In general recombination occurs in vivo between two homologous DNA molecules whereas in vitro any two DNA molecules irrespective of homology can be combined together.
What is a clone?
The term “clone” was derived from the Greek word – klon which means” a twig”. An aggregate of the asexually produced progeny of an individual or a group of replicas of all or part of a macromolecule (such as DNA or an antibody) constitute a clone. A clone is an individual grown from a single somatic cell of its parent and is genetically identical to it
Clone: a collection of molecules or cells, all identical to an original molecule or cell
What is Molecular cloning?
Molecular cloning involves isolation of desired DNA fragments and insertion into a carrier molecule which is capable of replicating by itself and also along with the insert and/or expressing the insert DNA when introduced into a suitable host.
Terminology:
Transduction is a process of DNA transfer from one bacterium to the other through the virus leading to recombination. Viruses that infect bacteria are called Bacteriophages. Bacreriophages adsorb onto the membranes of the bacteria, drill a pore into the cell membrane through which they introduce the DNA into the cytoplasm of the host bacteria, which gets circularized immediately and subsequently integrates into the host genome and remains dormant. This is called Prophage which can get replicated along with the chromosomal DNA for generations Upon sensing the favorable conditions, the DNA excises itself from the chromosomal DNA, replicates by rolling circle mode of DNA replicate to produce multiple DNA molecules which gets packaged into viral particles and are released by lysing the bacterial cell. These progeny bacteria when infect other bacteria, same procedure is repeated, but this time the introduces viral DNA has certain chromosomal regions from the previous bacterial host which undergo recombination with the chromosomal regions of the new host.
Transformation:
Transformation is a process of introducing exogenous DNA into cells without the aid of any biological agent. Introduction of DNA leads to the change in the genotype which further results in the change in the phenotype. The ability of an organism to respond to externals signals is called Competence. Some species of bacteria like Streptomyces species are naturally competent and undergo transformation naturally whereas others are made competent by physical or chemical methods. Physical methods include electroporation which utilizes electric current in short pulses to introduce the DNA. Chemicals like salts of divalent cations such as Calcium chloride, Lithium chloride etc are used to make the cells competent. Such cells when given a heat shock tend to allow DNA molecules into the cells.
Transformation is the most frequently employed method in molecular biology to introduce the plasmid vectors into the bacterial cells. Plasmid vectors carry genes encoding antibiotic resistance which can be used as selection markers to select the cells that have undergone transformation.
In general recombination occurs in vivo between two homologous DNA molecules whereas in vitro any two DNA molecules irrespective of homology can be combined together.
What is a clone?
The term “clone” was derived from the Greek word – klon which means” a twig”. An aggregate of the asexually produced progeny of an individual or a group of replicas of all or part of a macromolecule (such as DNA or an antibody) constitute a clone. A clone is an individual grown from a single somatic cell of its parent and is genetically identical to it
Clone: a collection of molecules or cells, all identical to an original molecule or cell
What is Molecular cloning?
Molecular cloning involves isolation of desired DNA fragments and insertion into a carrier molecule which is capable of replicating by itself and also along with the insert and/or expressing the insert DNA when introduced into a suitable host.
Terminology:
- Desired DNA molecule is called as insert DNA
- Carrier DNA molecule is called as vector
- The process of combining insert DNA with vector DNA is called as ligation.
- Transformation is the process of introducing exogenous DNA into a host cell and the method of introducing DNA into animal cell is called as transfection.
- Isolation of desired DNA fragments.
- Ligation with a vector to generate recombinant DNA
- Transformation into a suitable host.
Why to clone DNA?
- A particular gene can be isolated and its nucleotide sequence determined
- Control sequences of DNA can be identified & analyzed
- Protein/enzyme/RNA function can be investigated
- Mutations can be identified, e.g. gene defects related to specific diseases
- Organisms can be ‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc.
Methods of DNA Isolation:
The methods used for isolating a DNA fragment can be classified into two types; Non –specific and specific methods.
Nonspecific methods of DNA fragmentation involves Mechanical shearing of DNA like High speed stirring or centrifugation. Sonication involves usage of high frequency sound waves. Sudden release of pressure is applied in French press to fragment DNA. In all these methods process of cleavage is not specified and also the average size of the fragment to be obtained cannot be regulated.
Specific methods of DNA fragmentation employ restriction endonucleases:
Restriction enzymes
Enzymes present in bacteria which prevent or restrict bacteriophage infection are called as restriction enzymes. They restrict the infection by cleaving the viral DNA. Enzymes that cleave phosphodiester bonds present in nucleic acids are called as nucleases. There are two types of nucleases- Exonucleases and Endonucleases.
Exonucleases act only on linear DNA. They can act either from 5’ or 3’ end and cleave one nucleotide at a time. The products of cleavage are mononucleotides.
Endonucleases act at internal sites on DNA. The products of cleavage are oligonucleotides. They can act both on linear and circular DNA.
Restriction enzymes are site specific endonucleases that cleave DNA internally acting as molecular scissors.
Discovery of restriction modification system:
When bacteriophage lambda culture was infected onto E.coli c strain culture, the efficiency of plating (EOP) was found to be 1 in 10000 (10-4) (EOP is the ratio of number of bacterial cells getting infected by viral particles). When the progeny lambda phages obtained when cultured onto E.coli k strain culture or reinfected onto the same strain, EOP increased drastically by 10000 fold (EOP=1). This phenomenon is called as Modification.
During reinfection the viral DNA is regarded as self by the bacterial enzymes and hence it is not cleaved. Methylation of viral DNA by host enzymes prevents its cleavage. But when Lambda phages are infected onto E.coli k strain culture, the EOP decreases drastically by 10000 fold. This phenomenon is called Restriction. In this case, the lambdac DNA is regarded as foreign DNA and hence it is cleaved preventing the infection. Modification done by E.coli c starin is not recognized by E.coli k strain. Therefore modification is a temporary change and is host specific. Similarly lambdak phages can reinfect E.coli k strain successfully but fail to infect E.coli c strain.
There are 4 types of restriction modification systems-Type I, Type II, Type III, Type IIs
| Feature | I | II | III |
| Nature of Enzyme | Single polypeptide having three domains each one for recognition, cleavage and modification. | Two separate polypeptides one for recognition and cleavage other for modification | Single polypeptide with three domains |
| Recognition sequence | Asymmetric | Symmetric | Asymmetric |
| Cleavage site | 1000 base pairs away from recognition site | Within the recognition site | 24-26 base pairs way from recognition site |
| Requirements | ATP, Mg+2, s-Adenosyl Methioinine (sAM) | Mg+2 | ATP, Mg+2, sAM |
Advantages of Type II enzymes:
- As these contain separate restriction and modification subunits, by using either the Restriction or modification polypeptides both the activities can be performed as desired
- Symmetric recognition sequences yield sticky ends which are easy to ligate
- DNA fragments of defined length can be obtained as cleavage occurs within the recognition sequence
- The requirements for usage of these enzymes are less.
Due to the described advantages type II enzymes are mostly used in molecular cloning experiments, genomic DNA fragmentation for Hybridization, Molecular marker development.(RFLP, AFLP etc). Nevertheless Type I and type III enzymes are also used but rarely where cleavage is required away from recognition site like in SABRE experiments.
Classes of Type II Restriction enzymes:
They are sequence specific double stranded DNA endonucleases. These enzymes recognize sequences exhibiting Dyad symmetry (Palindromic sequences which exhibit 2 fold rotational symmetry.
Sticky end cutters:
Restriction enzymes after recognizing a sequence cleave both the strands of DNA at different points. This type of cleavage produces DNA fragments with single stranded overhangs. Because of the recognition sequences being symmetric, single stranded overhangs produced after cleavage are complimentary to each other. DNA molecules with such ends tend to associate with each other by means of complimentary base pairing between the overhang regions. Therefore these ends are called sticky ends or cohesive ends.
Enzymes which produce such type of ends are called sticky end cutters
Eg: EcoRI, Pst I, Bam HI, Hind III
Blunt end cutters:
Restriction enzymes after recognizing a sequence cleave both strands of DNA at same point. These enzymes do not produce any sticky ends or overhang regions. The ends produced by these enzymes are called as blunt ends or flush ends. Enzymes which produce such type of ends are called Blunt end cutters
Eg: Sma I, EcoRV.
Nomenclature of Restriction Enzyme:
The species name of the host organism is identified by the first letter of the genus name and the first two letters of the specific epithet to generate a three-letter abbreviation. This abbreviation is always written in italics.
Classes of Type II Restriction enzymes:
They are sequence specific double stranded DNA endonucleases. These enzymes recognize sequences exhibiting Dyad symmetry (Palindromic sequences which exhibit 2 fold rotational symmetry.
Sticky end cutters:
Restriction enzymes after recognizing a sequence cleave both the strands of DNA at different points. This type of cleavage produces DNA fragments with single stranded overhangs. Because of the recognition sequences being symmetric, single stranded overhangs produced after cleavage are complimentary to each other. DNA molecules with such ends tend to associate with each other by means of complimentary base pairing between the overhang regions. Therefore these ends are called sticky ends or cohesive ends.
Enzymes which produce such type of ends are called sticky end cutters
Eg: EcoRI, Pst I, Bam HI, Hind III
Blunt end cutters:
Restriction enzymes after recognizing a sequence cleave both strands of DNA at same point. These enzymes do not produce any sticky ends or overhang regions. The ends produced by these enzymes are called as blunt ends or flush ends. Enzymes which produce such type of ends are called Blunt end cutters
Eg: Sma I, EcoRV.
Nomenclature of Restriction Enzyme:
The species name of the host organism is identified by the first letter of the genus name and the first two letters of the specific epithet to generate a three-letter abbreviation. This abbreviation is always written in italics.
- Where a particular strain has been the source then this is identified.
- When a particular host strain has several different R-M systems, these are identified by roman numerals.
For example in EcoRI
E- First letter from the genus- Esherchia, written in Capital letter.
co – Next two letters from the species name – coli. written in small letters.
R- represents the Strain
I order of the enzyme isolated from the organism
E- First letter from the genus- Esherchia, written in Capital letter.
co – Next two letters from the species name – coli. written in small letters.
R- represents the Strain
I order of the enzyme isolated from the organism
| Enzyme | Enzyme source | Recognition sequence |
| SmaI | Serratia marcescens, 1st enzyme | CCCGGG |
| Hae III | Hemophilus aegyptius, 3rd enzyme | GGCC |
| Hind III | Hemophilus influenzae, strain d, 3rd enzyme | AAGCTT |
| BamHI | Bacillus amyloliquefaciens, strain H, 1st enzyme | GGATCC |
Isoschizomers :
In general DNA molecules when cleaved by two different restriction enzymes (Sticky end cutters) cannot be joined unless they produce same ends. But isoschizomers produce same overhangs after cleavage and are hence termed as ISO(=same) Schizo (=cut) Eg: Acc 651 and KpnI, BamHI and BglII
Neoschizomers:
Restriction enzymes that recognize the same recognition sequence but cleave the sequence in different ways to produce different types of ends are called Neoschizomers.
Eg: XmaI and Sma I
Recognition sites of restriction enzymes are mostly even numbered like 4bp, 6bp and 8bp and as the length of the recognition sequences increases probability of its occurrence decreases. Hence 8bp recognition sequences are rare. Rare cutters or 8-base cutters are employed to generate relatively small number of fragments with larger size in southern blotting experiments.
4-base cutters
MboI, DpnI, Sau3AI /GATC
MspI, HpaII C/CGG
AluI AG/CT
HaeIII GG/CC
TaiI ACGT/
6-base cutters
BglII A/GATCT
ClaI AT/CGAT
PvuII CAG/CTG
PvuI CGAT/CG
KpnI GGTAC/C
8-base cutters
NotI GC/GGCCGC
SbfI CCTGCA/GG
For instance, let us assume a DNA fragment from E.coli, of 10 kb which is digested with a 4 bp cutter. On an average, 40 fragments of approximately 250 bp each is obtained. As the recognition sequence is 4bp, the probability of finding such a sequence is once in 256bp.
[44=256bp (approximately 250bp) 10000/250 = 40.] Similarly, the probability of finding a sequence with 6bp recognition sequence is once in 46= 4096bp and the probability of finding a sequence with 8bp recognition sequence is once in 48= 65,546bp.
In mammals and other organisms whose G+C content is is either less tha or more than 50%, tthe probability cannot be applied as events should be unbiased (50%).Therefore, neither the average size of the fragment obtained nor the number of fragments obtained can be determined. This is evident from the below shown data.
| Enzyme | Target | Arabidopsis | Nematode | Drosophila | E. coli | Human |
| ApaI | GGGCCC | 25,000 | 40,000 | 6,000 | 15,000 | 2,000 |
| BamHI | GGATCC | 6,000 | 9,000 | 4,000 | 5,000 | 5,000 |
Published date : 21 Jun 2014 06:13PM
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