rDNA Technology_1238765467765321489.pptx

Recombinant DNA technology basics

1. rDNA Technology Urjita Sheth

2. rDNA Technology Recombinant DNA ( rDNA ) technology : is a field of molecular biology in which scientists "edit" DNA to form new synthetic molecules, which are often referred to as "chimeras". Recombinant DNA : is a form of artificial DNA that is engineered through the combination, insertion or replacement of one or more DNA strands, thereby combining DNA sequences that would not normally occur together HISTORY The Recombinant DNA technique was engineered by Stanley Norman Cohen and Herbert Boyer in 1973 They published their findings in a 1974 paper entitled " Construction of Biologically Functional Bacterial Plasmids in vitro ", in which they described a technique to isolate and amplify genes or DNA segments and insert them into another cell with precision, to create a transgenic.

3. Recombinant DNA technology was made possible by the discovery of REs by Werner Arber, Daniel Nathans, and Halminton Smith, for which they received the 1978 Nobel Prize in Medicine. Arber effectively showed geneticists how to "cut" DNA molecules ; soon to follow was the understanding that ligase could be used to "glue" them together Other milestones in the development of recombinant DNA include the collaboration between Stanley Cohen and Herbert Boyer in 1972 , and the 1976 founding of Genentech , the first company to work with rDNA in its drug development labs. In 1978 scientists were able to replicate Somatostatin , the protein that regulates human growth hormones.

4. GENE CLONING is essentially the insertion of the piece of foreign DNA into cell in such a way that the inserted DNA is replicated & inherited through the generations METHOD : GENE CLONING The Gene cloning procedure involves: Isolation of Gene Insertion of Gene into other piece of DNA called VECTOR Transfer of rVector into suitable host Selection of those cells which contain desired rVectors Growth of the selected host cell (can be indefinitely) Now, depending upon the application; If the ultimate aim is to get amplification of DNA fragment, for its genetic analysis; vector containing insert DNA is recovered & DNA fragment is separated But at present the main aim of these procedures is the expression of genes which code for the polypeptide which is either expensive or difficult to prepare.

5. rDNA products being used in human therapy Many human genes have been cloned in E. coli or in yeast. This has made it possible to produce unlimited amounts of human proteins in vitro. Cultured cells (E. coli, yeast, mammalian cells) transformed with a human gene are being used to manufacture more than 100 products for human therapy. Some examples: insulin for diabetics factor VIII for males suffering from hemophilia A factor IX for hemophilia B human growth hormone ( HGH ) erythropoietin ( EPO ) for treating anemia several interleukins granulocyte-macrophage colony-stimulating factor ( GM-CSF ) for stimulating the bone marrow after a bone marrow transplant granulocyte colony-stimulating factor ( G-CSF ) for stimulating neutrophil production, e.g., after chemotherapy and for mobilizing hematopoietic stem cells from the bone marrow into the blood.

6. tissue plasminogen activator ( TPA ) for dissolving blood clots adenosine deaminase ( ADA ) for treating some forms of severe combined immunodeficiency ( SCID ) parathyroid hormone several monoclonal antibodies hepatitis B surface antigen ( HBsAg ) to vaccinate against the hepatitis B virus C1 inhibitor ( C1INH ) used to treat hereditary angioneurotic edema (HANE) several types of interferons

7. TOOLS IN GENE CLONING GC utilizes certain biological products & biological agents for achieving its objects, these are known as GC tools or GE tools The basic tools of GC are: Restriction endonuclease DNA Ligase A suitable DNA molecule capable of self replication in selected host called vector A suitable organism that serves as the host for the propagation of rDNA i.e. vector containing DNA fragment to be cloned

8. ENZYMES IN GENE CLONING The gene cloning procedure initially developed mainly because of the two enzymes: Restriction endonuclease : to cut DNA at specific site. These are also known as molecular scissor. Ligase : to seal the cut or nicks that remain in the rDNA molecule. It is also referred to as molecular glue. ADDITIONAL ENZYMES & DNA SEQUENCES: Reverse transcriptase: is used for production of cDNA copies from mRNA Alkaline phosphatase : for removal of 5’-phosphate from DNA T4 poly ntd kinase : for addition of phosphate group to an end having a free 5’-OH SI nuclease: for removal of single stranded protruding ends ( both 3’ & 5’ extensions can be removed )

9. The klenow fragment of E.coli DNA polymerase I: to make protruding ends double stranded by extending the shorter strands Lambda exonuclease : for removal of ntds from 5’ ends ( an exonuclease removes one ntd from the end at a time from DNA, but it does not make internal cuts) E.coli exonuclease III: for removal of ntds from 3’ ends Exonuclease Bal 31: for making DNA fragments with blunt ended shorter from both its ends Terminal deoxynucleotidyl transferase : for addition of single stranded sequence at 3’ end to blunt ended fragments Linker & adapter oligo ntd sequences: for modification of cut ends of fragments LINKERS: used to create cohesive ends either on blunt ended DNA fragment or on fragment having unmatched or undefined sequences in their protruding ends ADAPTERS: are short chemically synthesized DNA double strands, which already have one or both sticky ends.

10. RESTRICTION ENDONUCLEASE Commonly known as molecular scissor Origin and function These are of bacterial origin Occurs naturally in bacteria as a weapon against the invading viruses as it cleaves the foreign DNA , thus Protect bacteria from bacteriophage infection as restrict virus replication Bacterium protects it’s own DNA by methylating those specific sequence motifs Named after the organism from which they were derived EcoRI from Escherichia coli BamHI from Bacillus amyloliquefaciens HpaI from Haemophilus influenza Sau3a from Staphyllococcus aureus Availability : Over 200 enzymes identified, many available commercially from biotechnology companies

11. Classes: Type I Cuts the DNA on both strands but at a non-specific location at varying distances from the particular sequence that is recognized by the restriction enzyme Therefore random/imprecise cuts Not very useful for rDNA applications Type II Cuts both strands of DNA within the particular sequence recognized by the restriction enzyme The class of enzyme most widely used is type II REs which possess both endonuclease & methylation activity It usually recognizes 4 or 6 ntd & normally cut the DNA sequences within these sequences Note that these sequences are pelindromic having rational symmetry means reads the same in the 5’  3’ direction on both strands

12. Some enzymes generate “blunt ends” (cut in middle) Others generate “sticky ends” (staggered cuts) Staggered cut will yield single stranded ends that are identical or complementary to each other Consequently, at low temperature there is tendency for single strand to associate each other by base pairing causing a reversible linking together with fragments which have been cut by same enzyme therefore such fragments are also known as sticky or cohesive ends.

13. ISOLATION OF DNA SEQUENCE TO BE CLONED SHORT GUN APPROACH This procedure is commonly employed when dealing with poorly characterized genome. It is the formation of gene library in which the entire genome is cut down randomly into large pieces of about 10 kbs or more in size using partial digestion with REs & then these pieces are cloned without any attempt to select particular sequences. Each of these fragment is linked to a vector & cloned into suitable host cell. When bacteria containing these fragments are plated out to give rise individual colonies, each colony will contain sample of genome & there is finite chance that desired gene will be present in any one of these sample provided. Enough colonies are produced, one can almost certain that at least one will contain desired gene. Here, the larger the genome & smaller the average fragment size, more colonies will have to be grown to find any particular gene.

14. Calculation shows that for 10 kbs of fragments, 1.5x10 3 colonies are needed for E.coli genome & 2x10 6 for Homo sapience . In later case, number of colonies required are unmanageable & therefore much larger fragments about 40 kbs in size must be cloned, but the large fragment require special vector for cloning. Inevitably the gene will come from only as a small part of entire genome [approximately 0.03 % in case of E.coli & 0.03x10-3 in case of mammalian gene] so a way must be found to identify the gene & pull it out from the other part of genome either before or after cloning This job is much easier if the corresponding mRNA will be available in the fairly pure form If the protein for which it codes is the major product of the cell, then mRNA will already be enriched for desired species

15. However the procedure used for enrichment of mRNA from the pool of gene are Size fractionation of mRNA In vitro translation of fractions Precipitation of translation product with specific Abs or To pass it from column containing oligo dT dimers which selectively forms complementation with polyA tail of mRNA & allow the mRNA to absorbed on column Methods of obtaining mRNA is depends upon the product & is most difficult step in the cloning, particularly if the gene product is not well characterized. After obtaining purified mRNA, it can be used to direct the synthesis of cDNA using enzyme Reverse transcriptase This results into DNA- RNA hybrid formation After removing RNA strand using alkali or RNase , the ssDNA can act as a template for the synthesis of second strand of cDNA using DNA polymerase enzyme This results in to dsDNA molecule

16. To obtain expression of cloned gene, it is necessary to understand the genetic organization of prokaryotes & eukaryotes. In eukaryotes, the coding regions of genes ( exons ) are generally broken by non-coding regions ( introns ) From the primary transcript from the nuclear gene the introns must be removed i.e. RNA processing to form mature translatable mRNA The RNA processing step cannot be carried out by prokaryotes so that a correct translation of cloned genes of eukaryotes is not possible Therefore, when bacteria are used as cloning hosts it is preferable to use synthetic DNA or complementary DNA

17. Synthetic DNA Nucleic acid chemistry has advanced so rapidly that it is now a routine process in many laboratory to synthesize a long DNA molecule of any desired ntd sequence In order to produce a specific DNA fragment containing coding region of protein, the DNA sequence is deduced by “reverse translation“ from the sequence of this protein using an automated DNA synthesis machine This machine will carry out each & every steps automatically It requires the supply of the reagents & type sequence of ntd to be produced Consequently, if amino acid sequence of polypeptide is known, than it may be easier to synthesize a gene & isolate the sequence similar to natural gene. Using such approach it is possible to produce DNA fragment of 20-100 bases, which can be connected together to make large synthesis Since the DNA synthesis procedure is completely under the control of the investigator ,it is also possible to produce sequences in which one or more bases have been changed making possible the production of highly specific mutations.

18. Examples of this technique are: The artificial synthesis of the gene for stomatostatin , a peptide hormone with 14 aa & the synthesis of the A&B chains of insulin, which were cloned &expressed in E.coli . PRODUCTION OF COMPLEMENTARY DNA Specific mRNA molecules isolated from mRNA rich cells or tissues are used as templates in vitro with the enzyme Reverse Transcriptase, produce cDNA . An example of the use of this method for cloning insulin gene from rats in Ecoli .

19. HYBRIDIZATION METHOD This method depends on the principle that mRNA forms complex with cDNA segment from which it has been transcribed This method is possible if protein encoding genes do not have introns First the total DNA from organism is isolated, than this dsDNA is treated with alkali or heat & converted into ssDNA by denaturation This strands are mixed with the mRNA transcribed by that genes. this complex is isolated & DNA is separated from RNA by RNase treatment the ssDNA thus obtained is converted into dsDNA by using DNA pol I enzyme this method is most suitable for isolating genes which exist in multiple copies mRNA pairs with cDNA protein to form DNA-RNA complex [hybrid] for ex: ribosomal genes

20. VECTORS A vector is a DNA molecule that has ability to replicate in appropriate host cell & into which the DNA fragment could be cloned is integrated for cloning & therefore a vector must have an origin of replication that amplify the fragment DNA incorporate into the host cell The choice of a vector depends on the design of the experimental system and how the cloned gene will be screened or utilized subsequently. There are two types of vectors Cloning vectors : All vectors used for propagation of DNA insert into suitable host are called cloning vectors Expression vector : when vector is designed for the expression i.e. production of proteins specified by DNA insert than it is termed as expression vector. Such vector contain control elements like promoter, operator, ribosomal binding sites ,etc.

21. An ideal vector contain following properties It should able to replicate autonomously when the objective of cloning is to obtain multiple copies of insert DNA fragment & the replication must be under relaxed condition, so that it can generate multiple copies of itself within the host cell Vector DNA should be ideally of less than 10kbs in size because larger DNA molecules are broken down during purification procedure & also large vector present difficulties during various manipulation required for gene manipulation It should be easy to isolate & purify means crude techniques of isolation should be applied It should be easily introduced into host cell It should have suitable marker gene that allows easy detection &/or selection of transformed host cells If the objective is DNA transfer than it should able to integrate DNA insert it carries into genome of host cell along with itself Vector should contain unique target side for as many as REs possible into which the DNA insert can be integrated without disrupting the essential function

22. The cells transformed with such vector molecule that contain DNA insert i.e. rVector should be identifiable or selectable from those transformed by only vector molecule When expression of DNA insert is desired , the vector should at least contain suitable control elements As all the above properties cannot be served by a single vector in nature, it is required to create useful vector by joining together segments performing specific function from two or more natural entities i.e. naturally available vectors

23. Requirements of a vector to serve as a carrier molecule Most vectors contain a prokaryotic origin of replication allowing maintenance in bacterial cells . Some vectors contain an additional eukaryotic origin of replication allowing autonomous, episomal replication in eukaryotic cells. Multiple unique cloning sites are often included for versatility and easier library construction. Antibiotic resistance genes and/or other selectable markers enable identification of cells that have acquired the vector construct. Some vectors contain inducible or tissue-specific promoters permitting controlled expression of introduced genes in transfected cells or transgenic animals. Modern vectors contain multi-functional elements designed to permit a combination of cloning, DNA sequencing, in vitro mutagenesis and transcription and episomal replication

24. Main types of vectors Plasmid: an extrachromosomal circular DNA molecule that autonomously replicates inside the bacterial cell; cloning limit: 100 to 10,000 base pairs or 0.1-10 kilobases (kb) Bacteriophage : derivatives of bacteriophage lambda; linear DNA molecules, whose region can be replaced with foreign DNA without disrupting its life cycle; cloning limit: 8-20 kb Cosmid : an extrachromosomal circular DNA molecule that combines features of plasmids and phage; cloning limit - 35-50 kb Bacterial artificial chromosome (BAC): based on bacterial mini-F plasmids. cloning limit: 75-300 kb Yeast artificial chromosome (YAC): an artificial chromosome that contains telomeres, origin of replication, a yeast centromere , and a selectable marker for identification in yeast cells; cloning limit: 100-1000 kb yeast 2 micron plasmid, retrovirus, baculovirus vector…….

25. CHOICE OF VECTOR Depends on : Nature of protocol or experiment Type of host cell to accommodate rDNA Prokaryotic Eukaryotic

26. Plasmid vector Covalently closed, circular, double stranded DNA molecules that occur naturally and replicate extrachromosomally in bacteria Plasmid can be considered as a suitable vector if it possess following properties: It should be easily isolated from the cell It should possess a single RE recognition site for one or more REs Insertion of linear molecule at one of these sites should not alter its replication properties It should have marker genes which allows easy detection & selection of transformed cells as rVector containing transformed cells It should be reintroduced into bacterial cell carrying plasmid with or without insert

27. pBR322 It is one of the most widely used vector & constructed by excising parts of naturally occurring plasmid using REs & rejoining them in a correct orientation & a series of manipulation which make it simple to use It is named after the scientist who discover this are BOLIVER & RODRIGNE The desirable features offered by resulting plasmid for cloning includes: Origin of replication It has been derived from naturally occurring plasmid col E1 The origin is particularly more useful because it is relaxed i.e. its replication is not linked to any part of chromosomal DNA &hence initiation of plasmid replication is more frequent than that of main chromosomal DNA Consequently multiple copies are accumulate within the cell so the process can be continued still further

28. 20 REs recognition sites this plasmid has 20 REs recognition sites so that any of the 20 enzymes can make cut in circular plasmid generating a single linear molecule to which the DNA fragment for cloning can easily attach Then this molecule can be reclosed to generate slightly enlarged circular plasmid Ampicilline & tetracycline resistance gene This gene of plasmid allows great flexibility in approach to selecting recombinant molecule

29. Selection of cells containing recombinant or non recombinant vector The presence of plasmid in a bacterium will confer resistance to antibiotic & such cells can be selected by growth on the medium containing either ampicilline or tetracycline Insertion inactivation phenomenon Some of the restriction sites are situated on the antibiotic resistance gene & so the insertion of the foreign DNA will lead to inactivation of the that gene For example : If the restriction recognition site/ insertion site is BamH1 that is present on the tetracycline resistance gene so the gene will be destroyed & the recombinant plasmid will allow cell to grow in presence of ampicilline but cannot protect the cell against tetracycline Screening for transformer containing vectors After transformation all the cells are allowed to grow on the medium containing ampicilline All the cell containing vectors / transformers are able to grow on ampicilline so colony appears on the ampicilline plate are of the host cell which has received plasmid or rPlasmid So to distinguish rPlasmid perform replica transfer of colonies from ampicilline plate to tetracycline plate

30. Those cells which unable to grow on tetracycline plates from ampicilline plate are recovered from ampicilline plate & are further characterised for the presence of insert DNA into plasmid

31. DNA Ligase DNA ligase were first time discovered by Martz & Davis in 1972 which can able to join two fragments of DNA either sticky or blunt There are two types of ligases are available from two different sources From E.coli From T4 phage ligase JOINING OF DNA The process of cloning not only depends on the availability of restriction enzymes & vector but also on our ability to join length of DNA together covalently If the DAN fragments to be joined have been cut using same REs to give identical cohesive ends ,they will tend to stick together if they collide & held by H bonding between complementary base pairs However, this association is only temporary owing to the weakness of bonds & the small no. of bases involved The linkage can be made permanent by using Ligase enzyme, which will form a phosphodiester bond between 5’,phosphoryl & 3’-hydroxil group. Thus joining 2 DNA molecules together or circularizing single linear molecule

32. The main source of Ligase is T4 phage & the enzyme required ATP to drive the ligation In order to stabilize 2 bps of cohesive end, the ligation is carried out at 4 ۟ -10 ۠ C temperature with long incubation time to allow for a low rate of enzyme activity at this temp. An imp. Feature of T4 DNA Ligase is its ability to join blunt end of DNA together, provided the conc. Of insert & enzyme are high enough the ligation depends on random collision of DNA having complementary ends The product of mixture of DNA fragments & linear vector molecule will include not only rVector carrying DNA insert but also, recircularized vector without insert length of DNA formed by joining of two or more fragments, circularized fragments by joining two or more vector molecules linked together,etc By careful choice of conc. of vector & DNA insert, the formation of plasmid containing single insert can be favoured , but unwanted product cannot be removed or eliminated completely For some purpose this type of ligation is adequate especially when subsequent step will select for recombinant product .

33. However there are ways of excerting more control over the product which can be formed, for example, If vector is treated with alkaline phosphatase after restriction, 5’phosphate group will be removed selectively Ligase will join 3’&5’ ends of DNA together if 5’ end is phosphorylated & so here, upon ligation with untreated DNA fragment link can only be formed b/n - fragment & fragment & - fragment & vector Thus preventing simple recirculrization of vector & increasing the yield of recombinant molecule Of course, fragment-vector linkage can only be made through one strand of DNA, but this is sufficient to hold the molecule together & the remaining two nicks will be eventually repaired by bacterial host after trasformation

34. USE OF LINKERS OR ADAPTERS But this is not always possible, so in such a cases any incompatible cohesive ends can be converted to blunt ends either by removing this protruding ends by SI nucleases or by filling single stranded ends by using DNA polymerase & all four ntd triphosphates Ideally both vector & insert DNA should be cut with same REs or with a pair to generate identical protruding ends i.e. schizoisomers The blunt ended molecules than can be linked using T4 Ligase However, this is not usually done, since the resulting recombinant molecule would probably not contain sites for restriction at the points where links have been made & so it would be difficult to remove the insert DNA at the point where links have been made & so it would be difficult to recover the insert DNA from vector after cloning Consequently, it is far more useful to carry out blunt ended ligation b/n DNA fragments & linkers which are chemically synthesized oligontds containing one or more restriction sites

35. If vector contain for example, Hind III cleavage site on one of its antibiotic resistance gene, than the DNA fragment after conversion to blunt ended form would be ligated to linkers containing Hind III sites Subsequent restriction to fragments plus linkers using Hind III would generate molecule which could be ligated to vector There are so many linkers as well as adapters available commercially so that it is now possible to overcome the almost all problems of incompatibility b/n vector & insert molecule.

36. HOMOPOLYMER TALING This technique can be used to join blunt ended molecules & has the advantage that only intermolecular bonds b/n vector & insert can occur A vector & insert are treated separately with terminal transferases & either dATP or dTTP , so that polyA tail are build up on the 3’-hydroxyl terminii of one hybrid molecule which can be used for transformation Upon mixing the complementary tails will results in stable hybrid molecules which can be used for transformation The disadvantage of this technique is that : it does not automatically create restriction site on either site of the inserted DNA & so recovery of insert may be difficult, but when dGTP & dCTP are used, similarity with the molecules which have been cut with the restriction enzyme Pst I because it generate Pst cleavage site & hence can also be used to recover insert from the plasmid after cloning.

37. SELECTIN OF HOST CELL The ideal host cell should possess following properties It should easy to transform It should support replication of rVector It should free from elements which interfere with replication of rDNA It should lack REs otherwise it rDNA will be cleaved down It should not have methylase activity otherwise it rDNA become resistant to REs & so recovery of insert DNA is very difficult It should be deficient in normal recombination functions so that insert is not altered by recombination event

38. SELECTION OF TRANSFROMED CELLS antibiotic resistance COLONY HYBRIDIZATION: It is possible to select & pick out the clones from the large number of colonies produced by transformation by colony hybridization It is largely depends upon the availability of radioactively labeled probes having sequence complementary to at least one part of desired DNA on a NA plate The colonies of transformed cells are replica plated on to a nitrocellulose filter & place it on surface of solid NA & incubate it to form colonies on filter The cells are lysed with alkali by treatment of filter with alkali, this will denature the DNA fixed to the filter by backing This way pattern of colony is replaced by identical pattern of bound denatured DNA The filter is then incubated with a solution of labeled probe which will hybridize to any bond DNA which contain sequences complimentary to the probe

39. If this is carried out at around 65C , hybridization will occur when there is an almost perfect match b/n the 2 nucleic acid sequences Following through washing to remove unbound probe, the hybridized probe is located by autoradiography of filter It is then possible to say which colonies contain DNA complimentary to probe & to pick those of from the master plate for further growth & analysis