RECOMBINANT DNA (RDNA) TECHNOLOGY

                   BY- ABHISHEKA G.(MSIWM013)

INTRODUCTION:

1.Recombinant DNA or RDNA technology is defined as the procedure of joining DNA molecules of two different species together and inserted into the host organism to produce a variety of new genetic combinations. This is also known as Genetic engineering.

2. The DNA fragments are selected from two different species and combined. This technique was developed by two scientists namely Boyer and Cohen in 1973.

3. The DNA molecule which is inserted into another DNA molecule is called a VECTOR. The recombinant vector is then introduced into a host cell where it replicates itself, and the new gene is produced. This is the basic principle behind Recombinant DNA technology.

TOOLS OF THE RECOMBINANT DNA TECHNOLOGY:

  1. Restriction endonucleases: These are used to cut DNA molecules at specific sequences into many smaller DNA fragments.
  2. Plasmids: These are extrachromosomal circular DNA present in the bacteria, which can replicate independently. During cloning, these plasmids carry drug resistance genes that are used for selection. Foreign DNA can be placed into a plasmid and it is replicated further.
  3. DNA ligase: This enzyme is used to join the two pieces of DNA together.
  4. Foreign DNA: This is also known as passenger DNA, which contains desired gene sequences.
  5. Vector: It is a vehicle used to insert the desired DNA into the host cell. Some of the vectors used are Plasmid DNA, Bacteriophage DNA, Yeast DNA, Viral DNA, Bacterial DNA, etc.

GOALS OF RDNA TECHNOLOGY:

  1. To isolate and characterize a gene or DNA from an organism.
  2. To eliminate undesirable phenotypic characters.
  3. To combine the needy and beneficial traits of two or more organisms.
  4. To make desired alterations in one or more isolated genes or DNA
  5. Inserting the altered genes or DNA into the host cell of another organism.
  6. To synthesize new genes using artificial methods.
  7. To alter the genome of the organism
  8. Understanding the diseases which transmit due to heredity.
  9. Understanding the treatment for heredity related disorders.
  10. To create new gene combinations.

PROCEDURE TO PREPARE RDNA:

1 Isolation of DNA from the organism: The cells are lysed using detergent mixtures, which creates pores in the plasma membrane. Then the mixture of cell contents is treated with protease and RNAase enzymes. The enzyme protease destroys the proteins present in the mixture and the enzyme RNAase destroys the RNA molecules present in the mixture. Then the mixture is centrifuged and the supernatant containing the DNA is transferred into a clean test tube and the DNA precipitated with the addition of ethanol.

2. Insertion of foreign genes into vectors: By using plasmid as a vector, isolated from the bacterial cell and treated with restriction enzymes and target DNA is obtained and it is placed into a vector to produce recombinant DNA.

3. Insertion of Recombinant DNA into host cell: The plasmid containing the foreign DNA is placed into a bacterial or host cell for multiplication.

4.Transformation: The vector is used as a vehicle to transport the gene to host cell, bacterium or other living cells are used as vectors. The vector is multiplied in the host cell and produces many identical copies, which are similar to both DNA and gene present in the DNA.

5. Cloning: After the division of the host cell the rDNA copies produced are transmitted to the progeny and further vector replication takes place in the progeny cell, with the continuous division of cells, a clone of identical host cells is formed. Each clone contains one or more copies of the rDNA molecule. Later the identical host cells are lysis and rDNA molecules are separated from the host cells.

APPLICATIONS OF RDNA TECHNOLOGY:

  1. This technology helps to grow crops which are resistant diseases and pesticides, crops of our choice, fruits, and flowers of attractive colors.
  2. This technique is employed in the production of artificial insulin and to deliver the drugs to target sites.
  3. Used in Molecular diagnosis of diseases.
  4. Used in Gene therapy.
  5. Employed in DNA fingerprinting.
  6. Used in the production of vaccines and pharmaceutical products.
  7. In the production of monoclonal antibodies.

DNA REPLICATION

DNA replication

Content

  • Introduction
  • Types of replication
  • Model of DNA replication
  • Enzymes involved in DNA replication

Introduction

  • It is the process of producing two identical copy of DNA from one original DNA molecule.
  • DNA replication is one of the most essential or important properties exhibited by DNA.
  • Evolution of all morphologically complex form of life is based on the replication.

Types of Replication

  • There are possibly three modes of replication:
  • Dispersive
  • Conservative
  • semi-conservative

Semi Conservative

  • In this mode of replication, two  old parental strands serve as template for synthesis of new daughters strands and each new DNA contains one strand from the parent and one from newly formed progeny.
  • The evidence of semi conservative replication of DNA was first presented by Meselson and Stahl in 1958.

Dispersive

  • The old DNA molecule would break into several pieces, each fragment would replicate and the old and new segment would be combined to yield two progeny DNA molecules; each progeny molecule contains  both old and new strands along its length.

Conservative

  • According to the conservative replication, the old parental strands remains together and newly formed daughter strands are also together.

Model of DNA Replication

  • The brief discussion of DNA replication model is as follows:
  • DNA applications initiate at certain unique and fixed point called origin.
  • There is unwinding of complementary strands of DNA duplex with the help of two enzymes DNA gyrase and DNA helicase, this process is called Melting.
  • Single strand binding protein are attached to two single stranded region so that they not join to form duplex.
  • Due to melting in the origin region it produces two Forks(Y), in the DNA duplex and one fork is located at the end of the melted region. Generally, both the fox are involved in replication and become Replication Fork.
  • After formation of replication fork ,Primase enzymes initiate transcription of the strand in the 3’-5’ direction. This results in the generation of 10-60 long primer RNA.
  • The free 3’-OH of the  RNA primer provide initiation point for DNA polymerase for the sequential addition of deoxyribonucleotide.
  • The replication of the second strand (5’-3’) DNA is discontinuous, Hence 3’-5’strand of DNA is termed as the Leading strand and 5’-3’ is termed as Lagging strand.
  • Lagging strand generates small polynucleotides fragment called Okazaki fragments, during replication. This fragment is about 1000-2000 nucleotides long in E.coli.

Enzymes Involved in the DNA Replication.

  • There are some important enzymes which are involved in the replication of DNA :
  • DNA polymerase
  • Primase
  • Polynucleotide ligase
  • Endonuclease
  • Helicase
  • Single stand binding protein (SSB).

DNA polymerase

  • This enzyme synthesizes new strand on a template DNA strand.
  • It is also known as DNA replicase.
  •  Its activity was first explained by Kornberg in 1956.
  • In prokaryotes, there are five types of DNA polymerase:
DNA polymeraseGeneFunctions
1polAMajor repair enzyme
2polBMinor repair enzyme
3polCDNA replication
4dinBSOS repair
5umuD2’CSOS repair

Primase

  • It involves in the synthesis of RNA primer, which are required for initiation of DNA replication. It is RNA polymerase that are used only to synthesize primer during replication.

Ligases

  • This involves in the formation of phophodiester linkage and joining of two newly formed DNA strands.

Endonucleases

  • It produces an internal cut in a DNA molecule, while Restriction Endonucleases are those that cuts at only specific site or sequences.

Single strand binding protein

  • It prevents from forming duplex by binding to single strand DNA.