👑 Enzymes

👉🏻 Among many, the two core techniques that enabled birth of modrn biotechnology are

1. Genetic Engineering
2. Maintenance of sterile (microbial contamination-free) ambience

Tools and Techniques of Genetic Engineering

• Genetic engineering involves cutting of desired segments of DNA and pasting of this DNA in a vector to produce a recombinant DNA (rDNA).
• The biological tools used in the synthesis of recombinant DNA include enzyme, vehicle or vector DNA, passenger DNA and host cells.

Enzymes

Lysing enzyme

• These enzymes are used for opening the cells to get DNA for genetic experiment.
• Bacterial cell wall is commonly dissolved with the help of lysozyme.

Cleaving enzyme

👉🏻 These enzymes are used for DNA molecules. Cleaving enzymes are of three types:

1. Exonucleases cut off nucleotides from 5’ or 3’ ends of DNA molecule.
2. Endonucleases break DNA duplex at any point except the ends.
3. Restriction Endonucleases cleave DNA duplex at specific points in such a way that they come to possess short single stranded free ends. These enzymes scan DNA molecules with a particular sequence, usually of four to six nucleotides. Once it finds this recognition sequence, it stops and cuts the strands. This is also known as enzyme digestion, i.e. AVA I, Bam HI, Eco RI, Hae III, Bgl II etc. For example, a restriction endonuclease ECOR-I (from Escherrichia coli) recognizes the base sequence GAATTC/CTTAAG in DNA duplex and cleaves its strands between G and A.

• Restriction enzymes are used in recombinant DNA technology because they can be used in vitro to recognize and cleave within specific DNA sequence typically consisting of 4 to 8 nucleotides. This specific 4 to 8 nucleotide sequence is called restriction site and usually palindromic, this means that the DNA sequence is the same when read in a 5’ to 3’ direction on both DNA strand. Eg. AND MADAM DNA
• As a result the DNA fragments produced by cleavage with these enzymes have short single stranded overhang at each end these kinds of ends are called sticky or cohesive ends because base pairing between them can stick the DNA molecule back together again.

Synthesizing enzymes

👉🏻 These enzymes are used to synthesize new strands of DNA, complementary to existing DNA or RNA template. They are of two types:

1. Reverse transcriptase helps in the synthesis of complementary DNA (cDNA) strands on RNA templates.
2. DNA polymerase help in the synthesis of complementary DNA (cDNA) strands on DNA templates.

Joining enzymes

• These enzymes help in joining the DNA fragments.
• For example DNA ligase form Escherichia coli is used to join DNA fragments.
• Joining enzymes are, therefore called molecular glues.

Alkaline phosphatases

• These enzymes cut off phosphate group form the 5’ end of linearized circular DNA and prevent its recircularization.

  

Vehicle DNA or Vector DNA

• The DNA used as carrier for transferring a fragment of foreign DNA into a suitable host is called vehicle or vector DNA.

Vectors for cloning genes in plants and animals

• Agrobacterium tumefaciens a pathogen of several dicot plants delivers a piece of DNA known as T-DNA to transform normal plant cells into a tumor.
• Similarly Retroviruses as animals have the ability to transform normal cells into cancerous cells.
• A better understanding of the art of delivering genes by pathogens in their eukaryotic hosts has generated knowledge to transform these tools of pathogen into useful vectors for delivering genes of interest to humans.
• The tumor inducing (Ti) plasmid of Agrobacterium tumefaciens has now been modified into a cloning vector which is no more pathogenic to the plants but is still able to use the mechanisms to deliver genes (disarmed) and are now used to deliver desirable genes into animal cells.
• So, once a gene or a DNA fragment has been ligated into a suitable vector it is transferred into a bacterial, plant or animal host (where A multiplies).

Some example of vectors

• Plasmid: They are extra chromosomal DNA segments found in bacteria which can replicate independently. Plasmids can be taken out of bacteria and made to combine with desired DNA segments by means of restriction enzyme and DNA ligase. A plasmid carrying DNA of another organism integrated with it, is known as recombinant plasmid or hybrid plasmid or chimeric plasmid.
• Virus: The DNA of certain viruses is also suitable for use as a vehicle DNA. Bacteriophage (bacterial virus) has been used to transfer gene for ß galactosidase from Escherichia coli to human cells. Lamda phage (λ Phasge) has been used for transferring lac genes of E. coli into haploid callus of tomato.

Passenger DNA

• It is the DNA which is transferred from one organism into another by combining it with the vehicle DNA.
• The passenger DNA can be complementary, synthetic or random.

Complementary DNA (cDNA)

• It is synthesized on mRNA template with the help of reverse transcriptase and necessary nucleotides.
• The DNA strand is then separated from the hybrid RNA-DNA complex by using alkali.
• Complementary DNA strand is then synthesized over the template of cDNA with the help of DNA polymerase.
• cDNA formed through reverse transcription is shorter than the actual or in vivo gene because of the absence of introns or non-coding regions.

Synthetic DNA (sDNA)

• It is synthesized with the help of DNA polymerase on DNA template.
• Kornberg (1961) synthesized first synthetic DNA from a mixture of deoxyribonucleotide triphosphates, DNA polymerase enzyme, metal ions and a segment of viral DNA.
• Khorana (1968) synthesized first artificial gene (DNA) without a template. They synthesized the gene coding for yeast alanine t-RNA which contained only 77 base pairs. However, it did not function in the living system. In 1979, Khorana was able to synthesize a functional tyrosine t-RNA gene of E. coli with 207 nucleotide pairs. Since then a number of genes has been synthesized artificially.
• Random DNA: It refers to small fragments formed by braking a chromosome with the help of restriction endonucleases.