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.

MOLECULAR BIOLOGY AND GENETICS

BY: SREELAKSHMI (MSIWM012)

Molecular biology is the study of the chemical and physical structure of biological macromolecules. Genetics is a branch of science dealing with the study of heredity and variation.

 Molecular Biology is an overlapping with other areas of biology and chemistry. It is the understanding of the interactions between DNA, RNA and protein. It is basically of two steps: transcription and translation. It is called Central Dogma. Synthesis of RNA from DNA is called transcription and synthesis of Protein and DNA translation.

TRANSCRIPTION

  • Transcription involves synthesis of new strand of nucleic acid complementary to a DNA template strand.
  • To transcribe a gene, RNA polymerase proceeds through a series of well-defined steps which are grouped into three phases- initiation, elongation and termination.
  • The bacterial core RNA polymerase can initiate transcription at any point on a DNA molecule.
  • RNA polymerase can initiate a new RNA chain on a DNA template and therefore do not require a primer.
  • The elongating polymerase is a processive machine that synthesize and proofreads RNA.
  • Ribonucleotides enters the active site and the added to are growing RNA chain.
  • Termination of transcription is activated by the presence of terminator sequence which results in the elongating polymerase to dissociate from the DNA and release the RNA chain.

TRANSCRIPTION

In eukaryotes, they have three different polymerases and several initiation factors.

TRANSLATION

Translation converts the genetic information present within the mRNA to a linear sequence of amino acids in proteins

The decoding of mRNAs into the language of proteins is composed of four components which are Trna,aminoacyl tRNA synthetasesand also ribosome.mRNa template provides the information that must be interpreted.Aminoacyl Trna synthetase couple amino acids to specific tRNAs that recognize the appropriate codon. The protein coding region of each mRNA have contiguous and non-overlapping string of codon called an open reading frame. Translation starts at 5’end and ends at 3’end.it starts with start codon and ends with a stop codon.AUG is usually a start codon, whereas UAG,UGA,UAA are stop codon.

GENETICS

Genetics is the study of hereditary and variation. The term was first introduced by W Bateson.

Gregor Mendel is known as the Father of Genetics.

Mendel was the first one who told that there are some factors which give you a particular phenotype (eye color, hair texture).The pioneering study on generics was by Mendel on pea plants. He looked at how the size, height, colur.He selected only pure breeds. From result of this experiment he came up with the hypothesis:

  • LAW OF SEGREGATION:

It is studied with the help of monohybrid cross. It states that the alleles of a given locus segregate into separate gametes. It is also called as law of purity.

MONOHYBRID CROSS

  • LAW OF INDEPENDENT ASSORTMENT:

It can be explained with the help of dihydrid cross. Mendel considered seed form and cotyledon color for the cross. This law states that the factors or alleles of each character assort or segregate independent of the factors of other character at the time of gamete formation and get randomly rearranged in the offspring.

DIHYBRID CROSS

POST MENDELIAN

Basics of genetics was given by Mendel, later studies provided information on various genetic interactions which also led to the studies on various genetic disorders.

COMPLEMENTARY GENES

If two genes present on different loci produce the same effect when present alone but interact to form a new trait when present together .They are called complimentary genes.

SUPPLEMENTARY GENES

They are pair of non-allelic genes one of which produces its effect independently in the dominant state.

LINKAGE

It is an exception of principle of independent assortment.

CHROMOSOME THEORY OF LINKAGE

It was given by Morgan and Castle. It states that

  • The genes which show linkage are situated in the same chromosome and remain bounded by chromosomes by material.So,they cannot be separated during the processes
  • The degree or strengthen of linkage depends upon the distance between the linked genes on the chromosomes, closely located genes show strong linkage.
  • Genes lie in linear order in the chromosome.

CROSSING OVER

It is one of the two exceptions of Mendel’s law of independent assortment. It produces new combinations or recombination of genes by interchanging of corresponding segments between non sisters chromatids of homologous chromosomes at prophase 1 of meiosis the non-sister chromatids in which exchange of segments has occurred are called cross overs.