BIOTECHNOLOGY

BIOTECHNOLOGY

It is a technique of using live organisms such as microbes, plant or animal cells or their components to produce products useful to humans

PRINCIPLES OF BIOTECHNOLOGY

The two important techniques that enable the development of modern biotechnology are –

GENETIC ENGINEERING

It is a technique of alternating chemistry of genetic material (DNA and RNA) and introduction of it  into host organisms and thus changing the phenotype of the host organism

CULTURE TECHNIQUES

It facilitates the growth and multiplication of the desired microbe in large quantities for the manufacture of products like vaccines, antibiotics, and enzymes

HYBRIDISATION

It multiplicates undesirable genes along with the desired genes

Genetic engineering isolate and introduce desirable genes without introducing undesirable genes into the target organism

 

ORIGIN OF REPLICATION

The specific sequence of DNA which is responsible for initiating replication

CLONING

It is the process of making multiple identical copies of at template DNA

PLASMID

It is a circular extra chromosomal DNA that is self replication

Plasmid  act as vectors to transfer the piece of DNA attached to it

PLASMID

A new circular autonomously replicating DNA is formed known as recombinant DNA

it replicates using the new host’s DNA polymerase enzyme and makes multiple copies

The basic steps in the genetic modification of an organism:

▪ Identification of desired DNA fragment

▪ Introduction of the desired DNA fragment into suitable host

▪ Maintaining foreign DNA in the host and its transfer to the progeny

TOOLS OF RECOMBINANT DNA TECHNOLOGY

RESTRICTION ENZYMES

  • They are enzymes that can cut DNA at specific fragments.

  • They are also called as “molecular scissors”.

  • The sequences at which they cut the DNA are specific for the restriction enzyme. They allow the desired gene to be cut and be introduced in specific locations in the vector or host DNA.

Restriction enzymes belong to of nuclease class of enzymes

They are of two types

Exonucleases

  • They cut DNA at the terminal end

Endonucleases

  • Endonucleases cut DNA at specific positions within the DNA

Recognition sequence

  • It is a specific sequence of six base pairs of DNA that can be recognised by restriction enzymes  as a site to cut the DNA

Palindromic nucleotide sequences

  • It is a sequence of base pairs in DNA that reads the same on the two strands when the orientation of reading is kept the same that is 5'→ 3' in both strands

  • Restriction enzymes recognize the palindrome sites of DNA.Restriction enzymes cut the strand of DNA a little away from the center of the palindrome sites but between the same two bases on the opposite strands leaving single stranded] overhanging stretches  at the ends

STICKY ENDS

  • These are single-stranded overhanging stretches on each strand formed due to the action of the restriction enzyme. They are called sticky ends as they form hydrogen bonds with their complementary cut counterparts.

LIGASE

  • Ligase is the enzymes that join two DNA fragments

  • Presence of sticky ends facilitate the joining of DNA 

VECTORS:

  • These are plasmids that are used to multiply and transfer the desired gene from one organism to the next

  • When the cloning vectors are multiplied in the host the linked piece of DNA is also multiplied to the number equal to the copy number of the vectors.

  • The following are the features that are required to facilitate cloning into a vector are-

  • Origin of replication (Ori)

  • Selectable Marker

  • Cloning sites

Origin of replication

  • It is the sequence of DNA from where replication starts

  • Foreign DNA when linked to this sequence, replicate within host cell

Selectable Marker

  • Selectable marker identifies and eliminate non-transformants and selectively permit the growth of the transformants

  • Transformation is a procedure through which recombinant DNA is introduced in a host bacterium.

  • The genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers for E. Coli which is lacked in normal E.Coli

 

Cloning sites-

  • Cloning sites are the recognition sites of the 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- ligation of a foreign DNA at the Bam HI site of the tetracycline resistance gene in the vector pBR322

  • Agrobacterium tumifaciens, a pathogen of several dicot plants deliver a piece of DNA known as ‘T-DNA’ and transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen

  • The plasmid of Agrobacterium tumifaciens is modified into a cloning vector which is non more pathogenic to the plants\. It delivers the desired genes into a variety of plants

  • Retrovirus delivers genes of interest in animals

  • Desired gene or a DNA fragment ligated into a suitable vector is transferred into a bacterial, plant or animal host (where it multiplies).

  • DNA is a hydrophilic molecule, it can't pass through cell membranes

  • In order to allow bacteria to take up the plasmid, the bacterial cells must be made ‘competent’ to take up DNA

  • This is done by treating them with a divalent cation, such as calcium, which creates the pores in the cell wall of bacteria.This is done by heat shock .In this method mixture of rDNA and host cell is incubated on ice followed by placing them briefly at 420C (heat shock) and then putting them back on the ice

  • This enables the entry of recombinant DNA into bacteria

Microinjection

  • Recombinant DNA is directly injected into the nucleus of the host cell

Biolistics or gene gun

  • Host cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA





 

ISOLATION OF DNA

  • DNA is the genetic material of all organisms

  • To isolate DNA the membrane need to be break down

  • Enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus breaks the membrane

  • Ribonucleases are used to remove the RNA whereas proteases are used to remove the proteins.

  • The pure DNA can be obtained through precipitation via ethanol.DNA appears as fine threads in the suspension

FRAGMENTATION OF DNA BY RESTRICTION ENDONUCLEASE

  • Purified DNA molecules is incubated  with the restriction enzyme for restriction enzyme digestion

SEPARATION AND ISOLATION OF DNA FRAGMENTS :

  • Gel electrophoresis is a technique that separate fragments of DNA obtained through an action of a restriction enzyme

  • In this  negatively charged DNA  molecules move towards the anode under an electric field through a medium

  • Agarose gel matrix is used. It is natural polymer obtained from seaweeds

  • The DNA fragments separate or resolve depending on their size as well as the pore size of the gel. The smaller DNA fragments are able to migrate farther than the larger DNA fragments

  • Separated DNA is stained to visualize the DNA.DNA is stained with a compound known as ethidium bromide and then exposed to UV light

  • DNA stained with EtBr fluoresces under UV.

Elution

  • It is a method of purification and extraction of DNA from the gel

  • They are the DNA molecules that can carry a foreign DNA segment and replicate inside the host cells

LIGATION OF THE DNA FRAGMENTS INTO VECTOR

Gene of interest’ is introduced into the vector with space and ligase is added to join the DNA to the vector.

This results in the preparation of recombinant DNA.

AMPLIFICATION OF GENE OF INTEREST USING PCR

Polymerase Chain Reaction

Is a technology used to amplify a single or a few copies of a Piece of DNA TO GENErate thousands to millions  of copies of a particular DNA sequence

For this sets of primers (small chemically synthesized oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase are required

INSERTION OF RECOMBINANT DNA INTO THE HOST CELL/ORGANISM

  • Insertion of recombinant DNA is into host cell or organism: Recipient cell is made competent to take up the recombinant DNA

OBTAINING THE FOREIGN GENE PRODUCT

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