Introduction to rDNA Tecchnology.pptx

Introduction to Genetic engineering & rDNA technology, Methodologies and enzymes involved in r-DNA technology

1. INTRODUCTION TO RDNA TECHNOLOGY Dr. S. Sivaranjani Assistant Professor Dept.of Biotechnology Bon Secours College for Women Thanjavur

2. RECOMBINANT DNA TECHNOLOGY A technique mainly used to change the phenotype of an organism (host) when a genetically altered vector is introduced and integrated into the genome of the organism. So, basically, this process involves the introduction of a foreign piece of DNA structure into the genome which contains our gene of interest. This gene which is introduced is the recombinant gene and the technique is called the recombinant DNA technology. The technology used for producing artificial DNA through the combination of different genetic materials (DNA) from different sources is referred to as Recombinant DNA Technology. Recombinant DNA technology is popularly known as genetic engineering.  The recombinant DNA technology emerged with the discovery of  restriction enzymes  in the year 1968 by Swiss microbiologist Werner Arber. This process involves the introduction of a foreign piece of DNA structure into the genome which contains our gene of interest. This gene which is introduced is the recombinant gene and the technique is called the recombinant DNA technology.

3. MINIMALIST AESTHETIC SLIDESHOW INFOGRAPHICS STEP-1 Isolation Of Genetic Material STEP-3 Amplifying The Gene Copies Through PCR STEP-5 Insertion Of RDNA Into Host STEP-2 Cutting The Genes At The Recognition Sites STEP-4 Ligation Of The DNA Molecules

4. Step-1.   Isolation of Genetic Material. The first and the initial step in Recombinant DNA technology is to isolate the desired DNA in its pure form i.e. free from other macromolecules. Step-2.Cutting the gene at the recognition sites. The restriction enzymes play a major role in determining the location at which the desired gene is inserted into the vector genome. These reactions are called ‘restriction enzyme digestions’. Step-3 .   Amplifying the gene copies through Polymerase chain reaction (PCR). It is a process to amplify a single copy of DNA into thousands to millions of copies once the proper gene of interest has been cut using restriction enzymes. Step-4 .   Ligation of DNA Molecules . In this step of Ligation, the joining of the two pieces – a cut fragment of DNA and the vector together with the help of the enzyme DNA ligase. Step-5 .   Insertion of Recombinant DNA Into Host. In this step, the recombinant DNA is introduced into a recipient host cell. This process is termed as Transformation. Once the recombinant DNA is inserted into the host cell, it gets multiplied and is expressed in the form of the manufactured protein under optimal conditions.

5. ENZYMES INVOLVED IN RDNA TECHNOLOGY DNA ligase Reverse transcriptase Restriction endonuclease Terminal transcriptase Nuclease DNA polymease Ribonuclease -H Alkaline phosphatase Polynucleotide kinase

6. DNA LIGASE DNA ligase is isolated from E.coli and Bacteriophage commercially and used in recombinant DNA technology. The enzyme DNA ligase joins the DNA fragments with cloning vector.

7. REVERSE TRANSCRIPTASE It is also called  RNA-directed DNA polymerase , an  enzyme   encoded  from the genetic material of  retroviruses  that catalyzes the transcription of  retrovirus   RNA  (ribonucleic acid) into  DNA  (deoxyribonucleic acid). This catalyzed transcription is the reverse process of normal cellular transcription of DNA into RNA, hence the names  reverse transcriptase  and  retrovirus . Reverse transcriptase is central to the infectious nature of retroviruses, several of which cause disease in humans, including human immunodeficiency virus ( HIV ). Reverse transcriptase is also a fundamental component of a laboratory technology known as reverse transcription-polymerase chain reaction (RT-PCR), a powerful tool used in research and in the  diagnosis  of diseases such as  cancer .

8. RESTRICTION ENDONUCLEASE Endonucleases can recognize specific base sequence (restriction site) within DNA or RNA molecule and cleave internal phosphodiester bonds within a DNA molecule. Example: EcoRI , Hind III, BamHI etc . These enzyme is isolated from wide variety of microorganisms. Endonuclease enzyme degrades foreign genome when enter inside microbial cell but the host cell own DNA is protected from its endonuclease by methylation of bases at restriction site. There are 3 types of restriction endonuclease: Type I Restriction endonuclease: It has both methylation and endonuclease activity. It require ATP to cut the DNA It cuts DNA about 1000bp away from its restriction site eg .  Eco KI Type II Restriction endonuclease: It does not require ATP to cut DNA It cuts DNA at restriction site itself eg .  Eco RI ,  Hind  III Type III Restriction endonuclease: It requires ATP to cut DNA  It cuts DNA about 25bp away from restriction site. eg .  Eco PI

9. Property Type I RE Type II RE Type III RE Abundance Less common than Type II Most common Rare Recognition site Cut both strands at a non- specific location > 1000 bp away from recognition site Cut both strands at a specific, usually palindromic recognition site (4-8 bp ) Cleavage of one strand, only 24-26 bp downstream of the 3´ recognition site Restriction and modification Single multifunctional enzyme Separate nuclease and methylase Separate enzymes sharing a common subunit DNA cleavage requirements Two recognition sites in any orientation Single recognition site Two recognition sites in a head-to-head orientation

10. TERMINAL TRANSCRIPTASE It is the enzyme that converts blunt end of DNA fragments into sticky end. If the restriction enzyme cuts DNA forming blunt ends, then efficiency of ligation is very low. So the enzyme terminal transferase converts bunt end into sticky end. Terminal transferase enzyme synthesize short sequence of complementary nucleotide at free ends of DNA, so that blunt end is converted into sticky end.

11. NUCLEASE The enzyme nucleases hydrolyses the phosphodiester bond on DNA strand creating  3’-OH  group and  5’-P  group. It usually cut DNA on either side of distortion caused by thymine dimers or intercalating agents The gap is filled by DNA polymerase and strand is joined by DNA ligase Nucelase are of two types; endonuclease and exonuclease

12. DNA POLYMERASE DNA polymerase is a complex enzyme which synthesize nucleotide complementary to template strand. It adds nucleotide to free 3′ OH end and help in elongation of strand It also helps to fill gap in double stranded DNA. DNA polymerase-I isolated from E. coli is commonly used in gene cloning Taq polymerase isolated from  Thermus aquaticus  is used in PCR

13. RIBONUCLEASE –H [RNASE H] Reverse transcriptases are enzymes composed of distinct domains that exhibit different biochemical activities. RNA-dependent DNA polymerase activity and  RNase H activity  are the predominant functions of reverse transcriptases , although depending on the source organisms there are variations in functions, including, for example, DNA-dependent DNA polymerase activity . RNase H cleaves the RNA template of the RNA:cDNA hybrid concurrently with polymerization.The RNase H activity is undesirable for  synthesis of long cDNAs  because the RNA template may be degraded before completion of full-length reverse transcription. The RNase H activity may also lower reverse transcription efficiency, presumably due to its competition with the polymerase activity of the enzyme.

14. ALKALINE PHOSPHATASE The enzyme Alkaline phosphatase helps in removal of terminal phosphate group from 5′ end It prevents self annealing of vector DNA soon after cut open by restriction endonuclease

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