Tools of Recombinant DNA Technology

Tools of Recombinant DNA Technology

 

The tools of recombinant DNA technology are essential for manipulating and combining genetic material to produce new genetic combinations. These tools include restriction enzymes, ligase enzymes, vectors, host organisms, and polymerase enzymes.

 

1. Restriction Enzymes

 

  • Restriction enzymes, also known as restriction endonucleases, are enzymes isolated from bacteria that cut DNA molecules at specific sequences.
  • They are proteins produced by bacteria that cleave the DNA at specific sites, known as restriction sites. These enzymes are crucial to certain laboratory methods, including recombinant DNA technology and genetic engineering.
  • Restriction enzymes protect the live bacteria from bacteriophages by recognizing and cleaving at the restriction sites of the bacteriophage and destroying its DNA.
  • In the 1960s, researchers such as Werner Arber, Hamilton Smith, and Daniel Nathans further investigated the phenomenon of restriction in bacteria. They found that certain strains of bacteria could recognize specific sequences of DNA and cut them into smaller fragments, effectively destroying the foreign DNA.
  • In 1968, Hamilton Smith and colleagues isolated the first restriction enzyme, called HindII, from the bacterium Haemophilus influenzae. They found that HindII recognizes and cuts at a particular site within a specific sequence of six base pairs.

Nomenclature:

The naming of restriction enzymes is standardized and follows a specific convention:-

  • The first letter of the enzyme's name refers to the genus of the organism from which it was first discovered, while the second and third letters refer to the species of the organism.
  • The genus and species names are written in italics, as per the convention for naming biological taxa.
  • For example, the first letter 'E' in EcoRI refers to the genus Escherichia, and the next two letters 'co' refer to the species coli. The 'RI' in EcoRI indicates the strain or serotype of the organism, while the Roman numeral 'I' indicates the order of discovery in that organism.

 

Restriction enzymes belong to a larger class of enzymes called Nucleases. Restriction enzymes are typically categorized into two main types based on their mode of action: endonucleases and exonucleases.

 

  1. Endonucleases: These enzymes cleave the DNA strands at specific recognition sequences, typically within the molecule rather than at the ends. They create breaks within the DNA molecule, hence the name "endonucleases." These breaks can be blunt-ended or staggered, depending on the enzyme.

  2. Exonucleases: In contrast, exonucleases degrade DNA from the ends of the molecule. They progressively remove nucleotides one at a time from either the 3' or 5' end of the DNA strand.

 

Action of Restriction Enzyme

 

(a) Recognition: Each restriction endonuclease enzyme recognizes a specific DNA sequence, typically between 4 to 8 base pairs long. These sequences are often palindromic, meaning they read the same forward and backward on complementary DNA strands.

  • In DNA, a palindromic sequence is one where reading from 5' to 3' on one strand is identical to reading from 5' to 3' on the complementary strand.
  • One common example of a palindromic nucleotide sequence is the recognition site for the restriction enzyme EcoRI, which has the sequence 5'-GAATTC-3'. When read in the 5' to 3' direction on one DNA strand, it reads "GAATTC", and when read in the 5' to 3' direction on the complementary DNA strand, it also reads "GAATTC". This sequence is palindromic because it reads the same forward and backward when considering complementary base pairing.
  • 5′ —— GAATTC —— 3′

    3′ —— CTTAAG —— 5′

(b) Binding: Once a restriction endonuclease locates its specific recognition sequence on a DNA molecule, it binds tightly to the DNA at or near that sequence.

(c) Cleavage: After binding, the restriction endonuclease cuts the DNA backbone at specific points within or near its recognition sequence. The cleavage can occur in several ways:

    • Blunt-ended cleavage: The enzyme cuts the DNA backbone straight across both strands, producing blunt ends.
    • Staggered cleavage (or sticky ends): The enzyme cuts the DNA backbone asymmetrically, leaving short single-stranded overhangs at the ends of the DNA fragments. These overhangs are complementary to each other and can easily base-pair with other DNA fragments cut by the same enzyme, facilitating DNA recombination. The stickiness of the strands facilities the action of the enzyme DNA ligase.

 

 

(d) Recombinant DNA molecules, which are made up of DNA from many sources or genomes, are created in genetic engineering by the application of restriction endonucleases.

(e) DNA ligases can be used to link DNA fragments that have been cut by the same restriction enzyme and have the same type of sticky ends.

 

 

 

Separation and Isolation of DNA Fragments

 

In gel electrophoresis, restriction DNA fragments are separated based on their size and charge. Here's a general overview of how it works:

 

  1. Preparation of DNA: DNA samples containing restriction fragments are prepared. These fragments are generated by digesting DNA with restriction enzymes, which cut the DNA at specific sequences.

  2. Loading onto Gel: The DNA samples are loaded into wells at one end of a gel matrix made of agarose or polyacrylamide.

  3. Application of Electric Field: When an electric current is applied across the gel, DNA fragments migrate through the gel towards the positive electrode. The smaller fragments move faster through the gel matrix than larger ones.

  4. Separation of Fragments: As the fragments move through the gel, they become separated according to their size. Smaller fragments travel farther down the gel, while larger ones remain closer to the starting point.

  5. Visualization: After electrophoresis is complete, the DNA fragments are visualized using a staining method, such as ethidium bromide or SYBR Safe, which binds to the DNA and fluoresces under ultraviolet (UV) light. This allows the bands representing the separated DNA fragments to be seen.

  6. Isolation: Once the desired DNA fragments are visualized, they can be separated and cut out from the gel and extracted from gel piece. This step is known as Elution.

 

 

 

2. Cloning Vectors

 

  • Cloning vectors are small DNA molecules used to transport foreign DNA into host cells for replicating and cloning purposes. These vectors have specific features that enable efficient cloning of foreign DNA fragments. 
  • Types of cloning vectors include plasmids, bacteriophages, phagemids, bacterial artificial chromosomes, yeast artificial chromosomes, cosmids, retroviral vectors, and human artificial chromosomes. Each type has specific advantages and applications in gene cloning and molecular biology research.
  • They have the ability to proliferate within bacterial cells without the influence of chromosomal DNA. Because of their large number per cell, bacteriophages have very high genomic copy numbers within bacterial cells.

 

To facilitate cloning into a vector, the following features are required:

 

  1. Origin of replication (ori): This is a specific sequence from where replication starts. When any piece of DNA is linked to this sequence, it can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA. Therefore, if one wants to recover many copies of the target DNA, it should be cloned in a vector whose origin supports high copy number.
  2. Selectable marker: In addition to the origin of replication, the vector requires a selectable marker, which helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants. Transformation is a procedure through which a piece of DNA is introduced in a host bacterium. Normally, genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline, kanamycin, etc., are considered useful selectable markers for E. coli.
  3. Cloning sites: In order to link the alien DNA, the vector needs to have very few, preferably single, cloning or recognition sites for the commonly used restriction enzymes. The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic-resistance genes. For example, a foreign DNA can be ligated at the BamH I site of tetracycline resistance gene in the vector pBR322. The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA but can still be selected out from the transformants growing on ampicillin containing medium. The recombinants will grow in ampicillin containing medium but not on that containing tetracycline. However, non-recombinants will grow on the medium containing both the antibiotics.

 

 

 

Insertional Inactivation

 

The insertional inactivation and blue-white selection method is a technique commonly used in molecular biology to identify and select for recombinant DNA molecules. Here's how it works:

 

  1. Insertional Inactivation: In this step, a DNA sequence (often a gene of interest) is inserted into a vector, which is a DNA molecule capable of replicating inside a host organism (such as a bacterium). This insertion disrupts the function of a particular gene within the vector, typically the lacZ gene.

  2. Blue-White Selection: The lacZ gene encodes an enzyme called beta-galactosidase, which is involved in the breakdown of lactose. Bacteria that have a functional lacZ gene produce this enzyme, which can convert a substrate called X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) into a blue-colored compound.

    • White Colonies: Bacteria that have a disrupted lacZ gene due to the insertion of foreign DNA (recombinant bacteria) cannot produce beta-galactosidase and thus cannot convert X-gal into the blue compound. These bacteria will produce white colonies when grown on a medium containing X-gal.

    • Blue Colonies: Bacteria that have an intact lacZ gene (non-recombinant bacteria) will produce beta-galactosidase and can convert X-gal into the blue compound, resulting in blue colonies on the X-gal medium.

 

 

By using this method, researchers can easily distinguish between bacterial colonies that contain the desired recombinant DNA (white colonies) from those that do not (blue colonies). This allows for the efficient selection of bacterial clones that have successfully incorporated the gene of interest into the vector.

 

Vectors for cloning genes in Plants and Animals 

 

  • Gene cloning in plants and animals can be achieved using various vectors, including Ti plasmid, retroviruses, and lambda phage. These vectors have specific features that allow for the convenient insertion or removal of DNA fragments, often using restriction enzymes and ligase.
  • Ti plasmid, found in Agrobacterium tumefaciens, is a natural vector for cloning genes in plants. It is used to cause tumors in plants and can be modified to remove the tumor-causing genes, allowing for the introduction of desirable genes into plant cells. This method has been effectively used for gene transfer in plants, as it carries sites for insertion of foreign genes intended to be transferred.
  • Retroviruses are used to deliver desirable genes into animal cells. They can integrate their genetic material into the host cell's DNA, allowing for the expression of the introduced gene.

 

3. Competent Host (For Transformation with Recombinant DNA)

 

  • DNA being a hydrophilic molecule, cannot pass through cell membranes, Hence, the bacteria should be made competent to accept the DNA molecules.
  • They are living organisms, such as bacteria or yeast, that have been modified or treated in such a way that they can uptake and express foreign DNA. The ability to uptake foreign DNA is known as competence, and it is facilitated by specific genes and proteins that enhance DNA uptake.
  • To make bacteria competent for DNA uptake, several methods can be used, including chemical and physical methods.
  • The chemical method involves treating the bacteria with a divalent cation, such as calcium ions, which increases the permeability of the cell wall, allowing for the uptake of recombinant DNA.  This process is often followed by a heat-shock step, in which bacterial cells are briefly exposed to a high temperature (typically around 42°C) followed by rapid cooling on ice. This thermal shock makes the cells more permeable to DNA.
  • The physical method involves the direct injection of recombinant DNA into the nucleus of the host, known as microinjection.
  • In plants, cells are bombarded with high velocity microparticles of gold or tungsten coated with DNA called as biolistics or gene gun method.
  • When disarmed pathogen vectors infect a cell, they pass recombinant DNA to the host.