Steps of r-dna technology

Recombinant DNA technology

1. STEPS OF R-DNA TECHNOLOGY By Dilip O. Morani Asst. Prof. Shri D.D. Vispute college of pharmacy & Research center, Panvel

2. Definition The deliberate modification in genetic material of organism by changing nucleic acid directly is called gene cloning and is accomplished by several methods which are collectively known as rDNA technology.

3. Steps Isolation of target DNA Insertion of target DNA into suitable vector Cloning vectors Isolation and identification of recombinant genes

4. A. Isolation of target DNA DNA fragments to be cloned are called DNA insert. Desired DNA inserts can be obtained from genomic library, cDNA libraries, chemical synthesis and amplification through PCR (Polymerase chain reaction). All these processes are possible only due to enzymes.

5. Enzymes acting on DNA Restriction enzyme Restriction endonuclease enzyme recognize and cut DNA strand at specific sequence called restriction site. 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. Existence of these enzymes was first postulated by Werner Arber in the early 1960s while studying bacterial viruses.

6. Continue… Restriction enzymes name is designated by a 3 letter abbreviation for the host organism. (e.g. Escherichia coli-Eco) A strain or type identified is written as subscript (e.g. Escherichia coli strain K-Eco K). Roman numerals are used to indicate different restriction-modification systems in a strain, when more than one enzyme is obtained from the same organism (e.g. Haemophilus influenzae, serotype d, enzyme III- Hind III).

7. Types of restriction enzymes There are 3 types of restriction enzymes. 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. These are complex and not used in gene cloning as their cleavage sites are non-specific. eg.   Eco K

8. Type II Restriction endonuclease It does not require ATP to cut DNA It cuts DNA at restriction site itself These are used for gene cloning studies giving rise to discrete DNA fragments of defined length and sequence. These are also used for restriction mapping and gene cloning. eg.   Eco RI,  Hind  III

9. Type III Restriction endonuclease: It requires ATP to cut DNA  It cuts DNA about 25bp away from restriction site. They are intermediate between type 1 and type 2. These enzymes are not used in gene cloning. eg.   Eco P1

10. Restriction enzyme-Palindrome Type II enzymes are most important and cut DNA molecules at specific sequences usually 4-8 bases. The sequences recognised are palindromes. In palindrome, base sequence in the second half of a DNA strand is the mirror image of sequence in the first half. In a palindrome with rotational symmetry, base sequence in the first half of one strand of DNA double helix is mirror image of second half of its complementary strand.

11. Restriction enzyme-sticky & blunt ends Some restriction enzymes make staggered cuts on the two DNA strands, leaving 2-4 nucleotides of one strand unpaired at each resulting end. These unpaired strands are called as cohesive or sticky ends. Certain type II restriction enzymes cleave both strands of DNA at the same base pairs but in the centre of recognition sequence. These DNA fragments are called flush ends or blunt ends.

12. 2. Alkaline phosphatase (AP) This group of enzymes removes the phosphate group (PO 2- ) from 5' terminus of the DNA molecule. It is active at alkaline pH, hence the name ‘alkaline phosphatase’. Commercially, it is obtained from three major sources, viz., E. coli (bacteria), calf intestine and arctic shrimp.

13. continue… Treatment of vector DNA with AP is important in cloning experiments, as removal of 5'phosphate prevents self-annealing of the digested vector and increases the possibility of ligating with the insert DNA fragment in the presence of ligase. Also, radiolabeled DNA probes are prepared by initially removing the5'PO 3 2- by AP treatment, followed by polynucleotide kinase treatment in the presence of radioactive phosphate.

14. 3. Reverse transcriptase (RT) Reverse transcriptase (RT) is a RNA dependent DNA polymerase found in RNA viruses also called as retroviruses. This enzyme is involved in the replication of retroviruses, where the RNA genome is first converted into DNA and then integrated into the host. RT uses mRNA template instead of DNA for synthesizing new DNA strand. The complementary DNA strand formed on the mRNA template is called the complementary DNA (cDNA). RT also shows RNAse H activity that degrades the RNA molecule from a DNA-RNA hybrid.

15. Continue… Formation of a double stranded cDNA from the mRNA molecule using RT finds applications in genetic engineering. The cDNA thus formed from any mRNA can be cloned in an expression vector and its respective protein can be made to express in large quantities.

16. 4. Nucleases Nucleases are enzymes that degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand. Nucleases can be broadly categorized into exonucleases and (ii) endonucleases.  Exonuclease removes the terminal nucleotide of the DNA molecule by breaking the phosphodiester bond, whereas endonuclease breaks the internal phosphodiester bond. Different types of exonucleases can be categorised on the basis of number of strands they degrade in a double stranded DNA molecule. An exonuclease named Bal31 is isolated from a marine bacterium Alteromonas espejiana . It is a Ca 2+ dependent enzyme that degrades the nucleotides from both the strands of dsDNA molecule.

17. Continue… Similar to exonucleases, endonucleases can also be categorised based on whether they act on single or double stranded DNA. S1 nuclease is an endonuclease that is isolated from the fungus Aspergillus oryzae . It is a heat stable enzyme that functions at high ionic strength, low pH and in the presence of Zn 2+ ions. It cleaves only single stranded DNA.

18. DNA polymerase DNA polymerases are enzymes that catalyze the synthesis of a new DNA strand from a pre-existing strand. The enzyme adds deoxyribonucleotides to the free 3’-OH of the chain undergoing elongation. The direction of synthesis is 5’-3’. It has three major requirements for its activity; (1) a template strand for which the enzyme synthesizes a complementary strand; (2) a primer with a free 3’-OH group that hybridizes with the template to form a double stranded region that initiates the polymerization and (3) a pool of all the four dNTPs that are used to synthesize the new DNA strand. In addition, some cofactors like Mg 2+ ions may be required in a buffer solution with correct pH for optimum activity.

19. Continue… Different types of DNA polymerases are used in recombinant DNA technology.   E. coli DNA Polymerase I Klenow Fragment Thermostable DNA Polymerase Reverse Transcriptase  

20. Terminal transferase This group of enzymes catalyzes the addition of one or more deoxyribonucleotides to the 3' terminus of the DNA molecule. The enzyme can work on both double stranded as well as single stranded DNA molecules without the need of any primers. The enzyme is obtained from calf thymus tissue.

21. Continue… The enzyme is used for labelling 3'ends of DNA. Also, it can be used for adding complementary homopolymer tails to DNA molecules.  Terminal transferase can add complementary homopolymeric chains at 3' ends of vector and insert. The two molecules are joined together due to transient base pairing and finally the discontinuities are sealed using DNA ligase.

22. Polynucleotide kinase (PNK) This group of enzymes perform a role completely opposite to the one performed by AP. PNK catalyzes the transfer of a phosphate group from ATP to the 5' terminus of the DNA molecule. This enzyme is obtained from E. coli infected with T4 phage.

23. DNA ligase DNA ligase catalyzes the formation of a phosphodiester bond between the 5' phosphate of one strand and the 3' hydroxyl group of another. In nature the function of DNA ligase is to repair single strand breaks (discontinuities) that arise as a result of DNA replication and/or recombination. In recombinant DNA technology, ligases catalyze the joining of DNA of interest called as ‘insert’, with the vector molecule and the reaction is known as ligation. The discovery of DNA ligases was equally important as that of restriction endonucleases in the development of recombinant DNA technology. For molecular cloning, the most commonly used DNA ligase is obtained from bacteriophage T4. T4 DNA ligase requires ATP as cofactor and Mg 2+ ions for its activity

24. B. Insertion of target DNA into suitable vector It can be performed when both target gene and vector are cut with same restriction enzyme to produce identical cohesive ends. Ends of DNA strands produced by restriction enzymes have to be modified for gene cloning. 5 phosphate group of vector DNA is removed by alkaline phosphatase treatment to prevent vector circulation during DNA insert integration. Cohesive ends are converted to blunt ends by removing protruding nucleotides using S1 nuclease, which degrades single stranded protruding DNA.

25. Continue… Type II restriction enzymes form fragments having sticky or blunt ends and such DNA fragments are not easily ligated with cloning vectors. New DNA sequences are created by inserting synthetic DNA fragments called linkers. Inserted DNA fragments with multiple recognition sequences for restriction endonucleases are called polylinkers. Linkers are short, chemically synthesized, ds oligonucleotides. Linkers are ligated to the blunt ends of DNA to be cloned by using T4 DNA ligase. These DNA are cut to generate fragments with sticky ends by using specific restriction enzymes.

26. Continue… Adaptors are short, chemically synthesized DNA double strands which can be used to link ends of two DNA molecules which have different sequences at their ends. Adaptor is used in the 5 hydroxyl form to prevent self polymerization. The foreign DNA plus ligated adaptors is phosphorylated at the 5 termini and ligated into the vector previously cut with Bam HI. Adaptors are used to develop sticky ends.

27. Continue… Homopolymer sequences are added to the foreign DNA fragments to be cloned and vector used by terminal transferase enzyme. Terminal transferase reaction is used to add tail to blunt ended DNA with protruding 3-hydroxyl terminus. Vectors and inserts are treated separately so that poly dA tails build on the 3-termini of one and poly dT tails on the other. Homopolymer tails form a stable, hybrid recombinant DNA in the absence of ligation.

28. Cloning vectors It is the central component of a g e n e cloning process. A small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the f r a gm e n t i s c a r r i e d o u t b y t r eatin g the v e ct o r a n d the f o r e i g n DN A w it h a restriction enzyme t h a t c r e a t e s the same overhang, then ligat i n g t he fragments together.

29. Characteristics of a cloning vector Ori (Origin of replication) is a specific sequence of nucleotide from where replication starts. It should have selectable marker gene. It should have restriction sites: a synthetic multiple cloning site (MCS) can be inserted into the vector. R e p l i c a t e in s id e t h e h o s t c e l l t o f o r m m u lti pl e c o p i e s o f the recombinant DNA molecule. Less than 10kb in size.

30. Origin of Replication: Allow the vector as well as the foreign DNA to amplify in the host cell Selectable Marker: Antibiotic resistance genes- Allow the host to grow on selective media; Can selectively amplify this specific vector in the host cell Multiple Cloning Sites: Allow insertion of foreign DNA

31. Types of Cloning Vectors Th e y all o w th e e x ogeno u s D N A t o b e i n s e r t e d , s t o r ed a nd manipulated mainly at DNA level. Types Plasmid vectors Cosmids YACs

32. Plasmid Vector Plasmid vectors are double-stranded, extra-chromosomal DNA molecules, circular, self-replicating. Advantages: Small, easy to handle Easy purification Straightforward selection strategies Useful for cloning small DNA fragments (< 10kbp) Disadvantages: – Less useful for cloning large DNA fragments (> 10kbp)

33. Continue… C on t a i n s a n o r i g i n o f r ep l i ca t ion , al l o w i n g f o r r e p lic a t ion independent of host’s genome. C on t a i n s S e lecti v e m a r k e r s : S ele c t io n o f c e l l s co n tainin g a plasmid twin antibiotic resistance blue-white screening Contains a multiple cloning site ( MCS ) Easy to be isolated from the host cell. Plasmids range in size from 1.0kb to 250kb e.g. pUC8 is 2.1 kb and TOL is 117 kb in size.

34. pBR322 It was one of the first vectors to be developed in 1977. Th e ‘ p ’ indic a t e s tha t i t i s p la s m i d , ‘ B R ’ indic a t e s B o li v a r a nd Rodriguez. ‘322’ distinguishes it from the other plasmids produced in the same laboratory e.g. pBR325, pBR327, pBR328. It is 4363bp in size i.e. less than 10kb I t c a r r i e s t w o s e t s o f a n t ibio t i c r e s i s t a nc e ge n e s i . e . e it h e r ampicillin or tetracycline can be used as a selectable marker.

35. Continue… Each of the marker genes carries unique restriction sites and insertion of DNA into these sites inactivates the specific marker site. e.g. insertion of new DNA with Pst1, Puv1, Ppa1 or Sca1 inactivates the amp R gene. It has a high copy number. They are about 15 molecules present in transformed cells but it can be increased to 1000 to 3000 by plasmid amplification in the presence of protein synthesis inhibitor i.e. chloramphenicol. The vector comprises DNA derived from three different naturally occurring plasmids: the amp R gene is from R1 plasmid, tet R from R6-5 plasmid and the ori gene from pMB1 plasmid.

36. R e p l i c a p l a t i ng : tr an s f e r o f th e co l o ni e s f r om o ne plate to another using absorbent pad or Velvet transfer of colonies +ampicillin + ampicillin + tetracycline these colonies have bacteria with recombinant plasmid

37. Screening bacteria by replica plating

38. FROM pBR322 to pUC pBR322 requires double screening pBR322 has limited number of restriction site For these reasons pUC (on the left) was engineered

39. pUC19- Lac selection plasmid It is 2686 bp in size and is one of the most popular E. coli cloning vectors. Derived from pBR322 in which only the ori and the amp R genes remain. The nucleotide sequence of amp R gene has been changed so that it no longer contains the unique restriction sites. The restriction sites are clustered into the lac Z’ gene. It has a high copy number of 500-700 molecules per cell even before amplification. The identification of the recombinant cells can be achieved by a single step process i.e. by plating onto agar medium containing ampicillin and X-gal.

40. Advantages of pUC over pBR322? Single step screening MCS increases the number of potential cloning strategies available X-gal (also abbreviated BCIG for bromo-chloro-indolyl-galactopyranoside) is an organic compound consisting of galactoside linked to indole . X - g a l i s c l e a v e d b y β - g a l a c t o s i da s e y i e l d i n g g a la c to s e a nd 5 - b ro m o - 4 - chloro-3-hydroxyindole.  The latter is then oxidized i n t o 5 , 5 '- d i b r o m o - 4 , 4 '- d i c h l o r o - i nd i g o , a n insoluble blue product. Thus, if X-gal and an inducer of β-galactosidase (usually IPTG ) is contained within an agar medium on a culture plate, colonies which have a functional lacZ gene can easily be distinguished.

41. A cosmid is a t yp e o f hybrid plasmid that contains a Lambda phage cos sequence. normal bacteriopage packaging size. Cosmids ( cos sites + plasmid = cosmids) DNA sequences a re originally from the lambda phage . They are often used as a cloning vector in genetic engineering . Cosmids can be used to build genomic libraries . They were first described by Collins and Hohn in 1978. Cosmids can contain 37 to 52 (normally 45) kb of DNA, limits based on the They can replicate as plasmids if they have a suitable origin of replication: for e xam p l e S V 4 o r i i n m a mm a l i a n c e l l s , C o l E 1 o r i f o r d o u b l e - s t ra n d e d DNA replication or f1 ori for single-stranded DNA replication in prokaryotes . C o s m ids

42. Continue… They frequently also contain a gene for selection such as antibiotic resistance so that the transformed cells can be identified by plating on a medium containing the antibiotic. Those cells which did not take up the cosmid would be unable to grow. Unlike plasmids, they can also be packaged in phage capsids , which allows foreign genes to be transferred into or between cells by transduction . Plasmids become unstable after a certain amount of DNA has been inserted into them, because their increased size is more conducive to recombination . To circumvent this, phage transduction is used instead. This is made possible by the cohesive ends , also known as cos sites. In this way, they are similar to using the lambda phage as a vector, except all the lambda genes have been deleted with the exception of the cos sequence.

43. Continue… F e a t u r e s o f bo t h p l a s m i d a nd lambda phage cloning vectors. Circular. Do not occur naturally Origin ( ori ) sequence for E. coli . Selectable marker, e.g. amp R . Restriction sites (for cloning). Contain Phage  (lambda) cos sites which permits packaging into  phage heads and therefore introduction to E. coli cells. Packaging only occurs with 37-52 kb f r ag m ent s - s e l e c t io n f o r l a r ge fragments Useful for 37-52 kb. Packaged DNA is inserted into cells and then replicates as a very large plasmid

44. Cloning in a cosmid Desired ligation P r o d u c t s- these are packaged

45. Cloning in a cosmid Instead of transformation, desired ligation products are packaged and then transfected into cells Selection for colonies, not screening of plaques (not infectious)

46. • • V e c t o r s t h a t e n a b l e a rt i f ici a l chr o m o s o m e s t o b e created and cloned into yeast. Based on the chromosome of Yeast Features: • • • • • • • • • CEN1, centromere sequence  segregation TEL, telomere sequences  extremity protection ARS1, autonomous replicating sequence  replication Selectable marker (amino acid dependence, etc.) on each arm. Amp ori, origin of replication for propagation in an E. coli host. Restriction sites (for DNA ligation). Acquiring 150kbp it acquires chromosome like features SUP4 gene, a suppressor tRNA gene which overcomes the effect of the ade-2 ochre mutation and restores wild-type activity, resulting in colorless colonies. The host cells are also designed to have recessive trp1 and ura3 alleles which can be complemented by the corresponding TRP1 and URA3 alleles in the vector, providing a selection system for identifying cells containing the YAC vector. Useful for cloning very large DNA fragments up to 500 kb; useful for very large DNA fragments. • • YACs: Y east A rtificial C hromosomes

47. DESADVANTAGES OF YAC • • • • Very fragile and prone to breakage, Unstable, with their foreign DNA inserts often being deleted Loss of the entire YAC during mitotic growth Difficult to separate the YAC from the other host chromosomes • The yield of DNA is not high • Chimaerism

48. Transformation and growth of cells Mandell & Higa (1979) developed procedure of transformation in bacterial cells. rDNA enter into suitable host cell for expression of foreign DNA. It is mainly introduced in E. Coli to select recombinant and obtain many copies of recombinant vector. Specific method is selected for transformation and depends on types of vectors and host cells. Main methods used for gene transfer (rDNA) into host cells are transformation and transfection.

49. Transformation Rplasmids are introduced into host cells (E. Coli). Plasmid DNA and host cells are mixed and pretreated with calcium chloride at low temperature to enter plasmid DNA (rDNA) into bacterial cell. Transformed bacterial cells are spread on surface of agar plates for growth

50. T ransfection Hybrid of transformation and infection. Lambda phage is used to transfer foreign DNA into E. coli. Vectors containing lambda phage cos sequences are in-vitro packaged into empty lambda phage heads and forms complete lambda particles. These phage particles are used to infect E. coli cells. Vectors are used to transform E. coli cells directly as naked DNA. Infection by phage particles containing DNA insert is more efficient than direct transformation. Apart from E. coli, Bacillus subtilis, yeasts and mammalian cells are commonly used as cloning hosts.

51. Host cells The hosts are the living systems or cells in which the carrier of rDNA molecule or vector can be propagated. Host cells can be prokaryotic or eukaryotic. Microorganisms are preferred as host cells, since They multiply faster compared to cells of higher organisms.

52. E. coli This was the first organism used in the DNA technology experiments. The major drawback is that it cannot perform post translational modifications.

53. Eukaryotic Hosts These are preferred to produce human proteins, since these have complex structure suitable to synthesize complex proteins. Mammalian cells possess the machinery to modify the protein to the active form.(post translational modifications) E.g., Tissue plasminogen activator

54. D. Isolation and identification of recombinant genes Objective is to isolate the cells that contain recombinant vector from non-transformed cells. Recombinant cells express the characters while non-recombinants do not express the characters. Different methods are used for selection of recombinants.

55. 1. Direct selection Cloned DNA itself codes for resistance to the antibiotic ampicillin ( amp R ) and the recombinants can be allowed to grow on minimal medium containing ampicillin. Such recombinants can grow on medium and form colonies that contain amp R gene on its plasmid vector.

56. 2. Hybrid arrested translation (HART) Portion of mixture (mRNA) is used for in vitro translation and serve as control. Remaining portion of mRNA mixture is subdivided and mixed with denatured rDNA molecule. Mixture is incubated under suitable conditions favouring annealing. DNA insert present in a given clone is hybridize with complementary mRNA. mRNA mixture is used for in vitro translation and resulting mixture of polypeptides is subjected to electrophoresis. Protein bands obtained in each sample are compared with those obtained from control mRNA. DNA insert causing absence of desired protein are identified and isolated.

57. 3. Hybrid selection Recombinant vectors are purified, denatured and fixed separately to a solid support (nitrocellulose filter discs). DNA attached to each disc hybridizes with its complementary mRNA. mRNA bound to each disc is isolated separately and used for in vitro translation. Resulting polypeptides are identified by electrophoresis. Identification of specific polypeptide may be facilitated by using antibodies specific to it. Antibodies may be used for western blotting or RNA blotting methods.

58. 4. Colony hybridization/nucleic acid hybridization Used to identify those bacterial colonies in a petri plate which contain specific DNA sequence. Bacterial colonies are replica placed or phage plaques are directly lifted on nitrocellulose filters. Filter disc is removed and put on blotting paper soaked with 0.5 N NaOH solution. Alkali diffuses into filters, lyses bacterial cells and denatures their DNA.

59. Continue… Disc is neutralised by tris (hydroxy methyl) amino methane HCl buffer by maintaining high concentrations of the salt. cDNA is fixed properly by baking at 80 c . Disc is incubated with a solution containing radioactive chemical labelled probe (p32) at suitable conditions. Probe hybridizes any bound DNA that contains sequences complementary to probe. Unhybridized probe is removed by washing. Colonies that develop positive x-ray image are compared with water plate and these colonies are picked up for further studies.

60. References Pharmaceutical biotechnology- Fundamentals and Applications by Prof. Chandrakant Kokare , Sixth edition-August 2018, Nirali Prakashan, page no. 10.1 to 10.28,

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