'rDNA notes' .pdf

Tools in Molecular Biology Ms. Geetanjali Harale Cloning Vectors Cloning vector is a small DNA molecule capable of self-replication inside the host cell. Cloning vector is used for replicating donor DNA fragment within host cell. Characteristics of a cloning vectors It...

Tools in Molecular Biology Ms. Geetanjali Harale Cloning Vectors Cloning vector is a small DNA molecule capable of self-replication inside the host cell. Cloning vector is used for replicating donor DNA fragment within host cell. Characteristics of a cloning vectors It must be small in size It must be self-replicating inside host cell It must possess restriction site for Restriction Endonuclease enzymes Introduction of donor DNA fragment must not interfere with replication property of the vector It must possess some marker gene such that it can be used for later identification of recombinant cell It must possess multiple cloning site Types of vectors based on cellular nature of the host cell 1. Prokaryotic Vectors: This comprises of all vectors for bacterial cells. 2. Eukaryotic Vectors: This comprises of all the vectors for yeast, animal and plant cells. Examples of cloning vectors Plasmids (pUC series) Cosmids Phagemids M13 Shuttle vectors YAC vectors yeast artificial chromosomes Expression vectors pET MACs mammalian artificial chromosomes Plasmid vector Plasmids are extra-chromosomal circular double stranded DNA replicating elements present in bacterial cells. Plasmids show the size ranging from 5.0 kb to 400 kb. Plasmids are inserted into bacterial cells by a process called transformation. Plasmids can accommodate an insert size of upto 10 kb DNA fragment. Example : naturally occurring plasmids are Ti plasmids, F-factors, R-factors, Co/E1 plasmid. PLASMID (PBR322) This was the first widely used, purpose built plasmid vector. pBR322 has a relatively small size of 4,363 bp. Cloning limit: 0.1-10 kb p’ indicates as a plasmid ‘BR’ identifies Boliver and Rodriguez, the two researchers who developed it. ‘322’ distinguishes those plasmids from others (like pBR 325, pBR 327, etc.) developed in the same laboratory. Marker gene: Ampicillin and Tetracycline resistant gene RSF2124 pSC101 pUC Vectors pUC are obtained by modifying the pBR322 vector. pUC vectors are smaller than pBR322 of being only ~2.7 kb. But comparatively they have a high copy number. 1. ‘p’ indicates the plasmid. 2. ‘UC’ stands for university of California where it was first developed by J. Messing et al. Selectable Marker: Ampicillin resistant gene. Phagemids Bacteriophages or phages are viruses that specifically infect bacteria. The phage particle attaches to the outer surface of the bacterium and injects its DNA into the cell. The phage DNA is then replicated inside the host and its genes are expressed to make phage capsid proteins and new phage particles are assembled and released from the bacterium. Phagemids Phage vectors can accommodate more DNA (upto 25 kb) than plasmids Two bacteriophages namely, Lambda (λ) and M13 have been commonly used for the construction of vectors for cloning in E. coli Phage M13 M13 is a filamentous bacteriophage of E. coli and contains a single-stranded circular DNA of 6407 bases. There is a segment of 507 nucleotide intergenic sequence (IS) which contains origin of replication (OR) This intergenic region can be manipulated for cloning without disrupting the origin of replication This region has only two restriction sites (Asa I and Ava Il), But it is not sufficient IS can be modified to introduce additional restriction sites Construction of M13 Based Vectors A series of vectors (M13 mp series) have been developed from this phage. These vectors have a polylinker with unique restriction enzyme sites in lacZ gene that complements host` The gene lacZ' is introduced into the wild types IS to get the M13 mp1 phages This produces blue plaques on X-gal agar plates The LacZ' gene does not have any restriction site Construction of M13 Based Vectors However, it has a hexanucleotide sequence, GGATTC near the start. If the second G residue is substituted by an A residue, this sequence becomes an Eco RI site: GAATTC This phage is called M13 mp2 The recombinant phages fail to produce blue plaques on X-gal agar, instead, they produce clear plaques M13 mp7 is a derivative of M13 mp2 When a polylinker is inserted into the Eco RI site of lasZ' gene; the M13 mp2 becomes M13 mp7 Construction of M13 Based Vectors The polylinker is designed in such a way that it does not inactivate the lacZ' gene. It has the Eco RI sticky ends and has restriction sites for Bam HI Sal I and Pst I. Thus M13 mp7 is a more complex vector with four possible insertional sites The recombinant M13 mp7 phage cannot produce blue plaques an X-gal agar plates due to insertional inactivation of lacZ' gene. They produce clear plaques M13-Plasmid Hybrid Vectors Hybrid vector contain component from both plasmids and phage chromosomes. These vectors replicate in E.coli as normal double-stranded plasmids until a helper phage is provided. After the addition of the “helper” phage, they switch to the phage mode of replication and package single strands of DNA in phage particles. The helper phage is a mutant that replicates its own DNA inefficiently, but provides viral replication enzymes and structural proteins for the production of plasmid DNA molecules that are packaged in phage coats Cosmids The cosmid vector is a combination of the plasmid and bacteriophage lambda. It is small (5-7 kb) circular DNA containing an origin for DNA replication (ori), selectable markers and restriction sites from plasmid plus a sequence from lambda needed for packaging the DNA (cos site). Cosmids may be used to clone large DNA molecules of up to 45 kb. YAC vectors Genetically engineered yeast minichromosomes. Accept foreign DNA inserts of 200-500 kb. Essential components Centromers (CEN), telomeres (TEL) and autonomous replicating sequence (ARS) of yeast for proliferation in the host cell. E. coli ori and selectable marker: you can grow the vector itself in E. coli ampr for selective amplification and markers such as TRP1 and URA3 for identifying cells containing the YAC vector. Recognition sites of restriction enzymes (e.g., EcoRI and BamHI) Auxotrophic markers URA3 The presence of the URA3 gene in yeast restores ODCase activity, facilitating growth on media not supplemented with uracil or uridine, thereby allowing selection for yeast carrying the gene. Positive selection is carried out by auxotrophic complementation of ura3 mutations, whereas highly discriminating negative selection is based on the specific inhibitor 5-fluoro-orotic acid (5-FOA) that prevents growth of the prototrophic strains but allows growth of the ura3 mutants. TRP1 The TRP1 gene encodes phosphoribosylanthranilate isomerase, an enzyme that catalyzes the third step in tryptophan biosynthesis.  SHUTTLE VECTORs Prokaryotic vector cannot exist & work in eukaryotic system because the systems of the two groups of org vary. Prokaryotic DNA lacks introns, while eukaryotic DNA contains introns. Therefore, several vectors have been constructed which may exist both in prokaryotes (E.coli) & in eukaryotic cells(yeast). Such vectors are known as SHUTTLE VECTORS.  What is a shuttle vector?? Defn :- Shuttle vector is “a vector (usually a plasmid) constructed so that it can propagate in two different species mainly a prokaryotes & a eukaryotes without any extra manipulation”. It possesses: i] two origins for replication (ori) i.e. 1 for prokaryotes & 1 for eukaryotes ii]two antibiotic resistance genes that act as a selectable markers. Importance of shuttle vector is that DNA cloned in one organism can be replicated in a second host without modifying the vector in any way to do so. Most of eukaryotic vectors are shuttle vectors. An example of shuttle vector is yeast episomal plasmid (YEP). Properties of a shuttle vector The vector must replicate in many org (e.g. bact,yeast & plants) to facilitate the isolation & characterization of genes. The vector must be easily recognized by selectable markers. The vector should be small in size to accommodate DNA inserts. Cloned genes should be easily detected. Useful quantities of vector must be easily obtained. The vector must be stable ,non-pathogenic & non-stress inducing. MODULE OF YEAST SHUTTLE VECTOR  Shuttle vector for E.coli and Streptomyces A shuttle vector designed to replicate in E.coli & Streptomyces has been constructed as follows i] Genes for DNA replication & methylenomycin A resistance are derived from Streptomyces plasmid ii] Replication module for maintenance in E.coli & a gene for antibiotic resistance are taken from E.coli plasmid.  Yeast Episomal Plasmid (YEp) YEp is 2 μm plasmid-based vector which replicates autonomously without integrating into yeast chromosomes. 2μm plasmid is 6 kb in size with high frequency for transformation. 2μm DNA consists of origin of replication & rep genes. This plasmid result in high frequency transformation i.e. about 2x104 transformants per μg DNA. Number of restriction sites is limited It is found in high copy number in the cells. It is moderately stable in cell. Expression vectors pET Advantages The T7 promoter is one of the strongest known promoters. It can produce a lot of protein. The PET plasmids have many common restriction sites, especially in front of the T7 promoter but also in other places. The very strong T7 promoter is regulated by the lac operator. In addition, the plasmids encode their own lac repressor which reduces the leakiness of the promoter. The pET plasmids have a medium copy number. (-20-25 per cell), which can be helpful because it prevents weird things from happening due to copy numbers that are too high or too low. It allows for the high expression level of the T7 promoter without overloading the cell with many copies of the plasmid in addition. Limitations Despite the strong selectivity of the T7 promoter for its phage encoded polymerase, residual "leaky" expression of very toxic proteins from the basic pET constructs can be sometimes lethal to the cell Applications The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli. The PET System provides six possible vector host combinations that enable tuning of basal expression levels to optimize target gene expression These options are necessary because no single strategy or condition is suitable for every target protein. 37  What are restriction endonucleases? Also called Restriction enzymes Molecular scissors that cut double stranded DNA molecules at specific points. Found naturally in a wide variety of prokaryotes An important tool for manipulating DNA. Named as host-induced or host-controlled. Discovery Arbor and Dussoix in 1962 discovered that certain bacteria contain endonucleases which have the ability to cleave DNA. In 1970 Smith and colleagues purified and characterized the cleavage site of a Restriction Enzyme. Werner Arbor, Hamilton Smith and Daniel Nathans shared the 1978 Nobel prize for Medicine and Physiology for their discovery of Restriction Enzymes. 38 Biological Role Most bacteria use Restriction Enzymes as a defense against bacteriophages. (Restriction endonucleases RESTRICT viruses) Restriction enzymes prevent the replication of the phage by cleaving its DNA at specific sites. The specific DNA sequence is called recognition sequence (Endo (inside), nuclease (cuts nucleic acid) 39  Nomenclature Smith and Nathans (1973) proposed enzyme naming scheme Three-letter acronym for each enzyme derived from the source organism First letter from genus Next two letters represent species Additional letter or number represent the strain or serotypes Example: EcoR1 Genus: Escherichia Species: coli Strain: R Order discovered: 1 40  EcoRI – Escherichia coli strain R, 1st enzyme BamHI st – Bacillus amyloliquefaciens strain H, 1 enzyme DpnI – Diplococcus pneumoniae, 1st enzyme HindIII – Haemophilus influenzae, strain D, 3rd enzyme BglII – Bacillus globigii, 2nd enzyme PstI – Providencia stuartii 164, 1st enzyme Sau3AI – Staphylococcus aureus strain 3A, 1st enzyme KpnI – Klebsiella pneumoniae, 1st enzyme  Recognition Sequences/Sites Each restriction enzyme always cuts at the same recognition sequence. Produce the same gel banding pattern (fingerprint) Recognition sites have two fold rotational symmetry (palindromic) For example MADAM 5’-GGATC Bam H1 site: C-3’ 3’-CCTAG G-5’ Palindrome-a word, verse, or sentence that reads the 42 same Palindrome Sequences Examples of Palindromes: Cuts usually occurs at a palindromic sequence Don't nod Dogma: I am God SmaI: produces blunt ends Never odd or even Too bad – I hid a boot 5´ CCCGGG 3´ Rats live on no evil star 3´ GGGCCC 5´ No trace; not one carton Was it Eliot's toilet I saw? EcoRI: produces sticky ends Murder for a jar of red rum 5´ GAATTC 3´ Some men interpret nine memos 3´ CTTAAG 5´ Campus Motto: Bottoms up, Mac Go deliver a dare, vile dog! Madam, in Eden I'm Adam Oozy rat in a sanitary  Mechanism of Action Restriction Endonuclease scan the length of the DNA, binds to the DNA molecule when it recognizes a specific sequence and makes one cut in each of the sugar phosphate backbones of the double helix – by hydrolyzing the phoshphodiester bond (between the 3’ O atom and the P atom is broken). Mg2+ is required for the catalytic activity of the enzyme. 44  Recognition sites May be between 4 and 12 bases long. Enzymes are often termed ‘four cutters’, ‘six cutters’ etc. based on the length of the site Usually pallindromic: reason for success. The enzyme attaches to BOTH strands and cuts them. In some cases not every base is constant The cut site may be centered or off-center: The former produce what are termed ‘blunt ends’ and the enzymes are termed ‘blunt cutters’ Most produce a ‘staggered cut’ that leaves ‘sticky ends’ 45 -P 3’ OHOH 5’ 3’- Sticky End Cutters Most restriction enzymes make staggered cuts Staggered cuts produce single stranded “sticky-ends” DNA from different sources can be spliced easily because of sticky-end overhangs. HindII I E co R I 46  Blunt End Cutters Some restriction enzymes cut DNA at opposite base They leave blunt ended DNA fragments These are called blunt end cutters Al uI H ae II I Sticky ends or blunt ends can be used to join DNA fragments. Sticky ends are more cohesive compared 47 to blunt ends. 48  5’ OVERHANGS The enzyme cuts asymmetrically within the recognition site such that a short single-stranded segment extends from the 5' ends. Bam HI cuts in this manner. 3’ OVERHANGS Asymmetrical cutting within the recognition site, but the result is a single-stranded overhang from the two 3' ends. KpnI cuts in this manner. Isoschizomers and Neochischizomers Restriction enzymes that have the same recognition sequence as well as the same cleavage site are Isoschizomers. Restriction enzymes that have the same recognition sequence but cleave the DNA at a different site within that sequence are Neochizomers. E.g: SmaI and XmaI CCCGGG CCCGGG GGGCCC GGGCCC Xma I Sma I 50 51  Few Restriction Enzymes Target sequence Enzyme Organism from which derived (cut at *) 5' -->3' Bam HI Bacillus amyloliquefaciens G* G A T C C Eco RI Escherichia coli RY 13 G* A A T T C Hind III Haemophilus influenzae Rd A* A G C T T Mbo I Moraxella bovis *G A T C Pst I Providencia stuartii CTGCA*G Sma I Serratia marcescens CCC*GGG Taq I Thermophilus aquaticus T*CGA Xma I Xanthamonas malvacearum C*CCGGG  Why don’t bacteria destroy their own DNA with their restriction enzymes? Methylation - The host DNA is protected by Methylases which add methyl groups (CH3) to adenine or cytosine bases within the recognition site thereby modifying the site and protecting the DNA. 53  Discovery Typically bacteriophage have a single host they can infect, or a small number of related bacteria: the ‘host range’ In the 50’s and 60’s it was noticed that occasionally a virus could become active in a new strain but would no longer be effective in the original host This change in host range seemed to be associated with specific bases in the viral DNA being methylated: something that would have to be done by the host Methylases In the late 60’s Werner Arber and Stewart Linn discovered that most bacteria contained a class of enzymes termed methylases. These could recognize a specific sequence of DNA and add a methyl group onto certain bases, most often an adenine or cytosine. The presence of these methyl groups on the viral DNA seemed to be the cause of their ability to grow in some strains of bacteria, but not others. 54  R-M System Restriction-modification (R-M) system Endonuclease activity: cuts foreign DNA at the recognition site Methyltransferase activity: protects host DNA from cleavage by the restriction enzyme. Methylate one of the bases in each strand Obviously if a bacterium is going to have a restriction enzyme, it also needs the methylase (aka modification enzyme), otherwise it will destroy its own DNA Restriction enzyme and its cognate modification system constitute the R-M system 55  Classification – restriction endonuclease Highly heterogeneous Evolved independently rather than diverging form a common ancestor Broadly classified into four Types Restriction enzymes are classified based on recognition sequence and methylation pattern 56  Type I These were the first to be discovered. Multi-subunit proteins which function as a single protein complex, though coded separately Contain two R (restriction) subunits, two M (methylation) subunits and one S (specificity) subunit Cleave DNA at random length from recognition site The binding is at a specific recognition site, which can be methylated and then the DNA loops back. The cut site may be as far as 1000 bases from the recognition site Each is used just once, then inactivated 57 Type III Large enzymes Combination restriction-and-modification Cleave outside of their recognition sequences Require two recognition sequences in opposite orientations within the same DNA molecule No commercial use or availability 58 Type IV Cleave only modified DNA (methylated, hydroxymethylated and glucosyl-hydroxymethylated bases) Recognition sequences have not been well defined Cleavage takes place ~30 bp away from one of the sites. Sequence similarity suggests many such systems in other bacteria and archaea. 59  Type II Most useful for gene analysis and cloning The nuclease and methylase are separate molecules, but recognize the same base sequence The nuclease will not attach if the site is methylated The cut site is within the recognition site: therefore a given enzyme will always cut in the same way, unlike Type I nucleases More than 3500 REs Recognize 4-8 bp sequences Need Mg 2+ as cofactor Cut in close proximity of the recognition site Homodimers ATP hydrolysis is not required 60  EcoK I EcoA I CfrA I EcoP I Hinf III EcoP15 I Type II RE are most useful for cloning They make double-stranded cuts They generate predictable ends 61 62  STAR ACTIVITY - RE The precise specificity of restriction enzymes for target sequences is considered as their most interesting characteristic. Although all restriction enzymes bind DNA nonspecifically, under optimal conditions the difference in cleavage rates at the cognate site and the next-best site (single base substitution) is very high. For example, the rate difference for EcoR I at its cognate site (5'-GAATTC-3') and next-best site (5'-TAATTC-3') is of the order of 105. Similarly, for EcoR V, cleavage at its cognate site (5'-GATATC-3') is 106 times faster than at the next-best site (5'-GTTATC-3‘) 63  However, under non optimal conditions, the differences in cleavage rates between cognate and next-best sites change dramatically for many enzymes. This loss of fidelity or increase in cleavage at sites similar to the cognate site is commonly referred to as star activity. A number of reaction parameters can increase the rate of cleavage at star sites relative to cognate sites. These include pH, type of ions present, ionic strength, metal cofactors other than Mg2+, high DNA : enzyme ratios and the presence of volume excluders (glycerol, ethylene glycol, etc.) In conjunction with this increase in star activity, cleavage rates at the cognate site generally decrease. 64  Joining DNA Molecules There are currently three methods for joining DNA fragments in vitro. The ability of DNA ligase to join covalently the annealed cohesive ends produced by certain restriction enzymes. The ability of DNA ligase from phage T4-infected E. coli to catalyse the formation of phosphodiester bonds between blunt-ended fragments. Utilizes the enzyme terminal deoxynucleotidyltransferase to synthesize homopolymeric 3′ single-stranded tails at the ends of fragments. 65  DNA Ligase (Joining) DNA ligases closes nicks in the phosphodiester backbone of DNA Biologically essential for joining of Okazaki fragments during DNA replication for completing short patches of DNA during DNA repair 66  Classes of DNA ligase Two classes Cofactor Found In NAD+ Only in bacteria (E. coli, B. subtilis) ATP Eukaryote, Viruses & Bacteriophages (T4, T7) The mechanism of DNA ligase is to form covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor"). In case of T4 ATP is required for the ligase reaction, which proceeds in three steps: adenylation (addition of AMP) of a lysine residue in the active center of the enzyme (forming a covalent ligase-(lysyl-N)-AMP intermediate, and pyrophosphate is released; transfer of the AMP to the 5' phosphate of the so-called donor (DNA-intermediate (AppDNA), forming pyrophosphate bond; a nucleophilic attack of the 3’-OH end so-called acceptor of the DNA on the AppDNA creates a phosphodiester bond, which seals the two DNA ends. Ligase will also work with blunt ends, although higher enzyme concentrations and different reaction conditions are required. 67 68 The ligation reaction 69  Sticky-end ligation When termini created by a restriction endonuclease that creates cohesive ends associate, the joint has nicks a few base pairs apart in opposite strands. DNA ligase can then repair these nicks to form an intact duplex. The optimum temperature for ligation of nicked DNA is 37°C, but at this temperature the hydrogen bonded join between the sticky ends is unstable. EcoRI-generated termini associate through only four AT base pairs and these are not sufficient to resist thermal disruption at such a high temperature. The optimum temperature for ligating the cohesive termini is therefore a compromise between the rate of enzyme action and association of the termini, and has been found experimentally to be in the range 4–15°C 70  T4 DNA Ligase The DNA ligase from bacteriophage T4 is the ligase most-commonly used in laboratory. It can ligate cohesive ends of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids. It can also ligate blunt-ended DNA with much greater efficiency than E. coli DNA ligase but with higher conc. It has an absolute requirement for ATP as a cofactor with Mg++ and sulfhydryl reagents (DTT, mercaptoethanol). T4 DNA ligase is a single polypeptide of M.W. 68 Kda E. coli DNA Ligase Used in Molecular biology NAD+ cofactor for ligase enzyme Cannot generally blunt ends but in presence of Ficoll & PEG it shows ligation 71  Blunt-end ligation The E. coli DNA ligase will not catalyze blunt ligation except under special reaction conditions of macromolecular crowding Blunt ligation is most usefully applied to joining blunt-ended fragments via Linker molecules Adaptor molecules Homopolymer tailing 72  Blunt end ligation via Linker Molecule A decameric linker molecule containing an EcoRI target site is joined by T4 DNA ligase to both ends of foreign DNA. Cohesive ends are then generated by EcoRI. This DNA can than be incorporated into a vector that has been treated with the same restriction endonuclease. 73  Blunt end ligation via Adaptor Molecule A synthetic adaptor molecule is ligated to the foreign DNA. The 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 BamHI. 74  Factors affecting Rate of DNA Ligation DNA conc. – high conc. results inter-molecular ligation whereas low conc. in intra-molecular Ligase conc. – higher conc., faster rate of ligation. Blunt-end ligation less efficient than sticky-end, so higher conc. of ligase used in blunt-end. Temp. – Optimum is 370 C & depends on Tm of ends of DNA Buffer Composition – Amt. of cations effect. Std buffer used to minimize ionic effect 75 Factors affecting Rate of DNA Ligation Uses DNA ligases are used in molecular biology To join DNA fragments with blunt or sticky ends such as those generated by restriction enzyme digestion Add linkers or adaptors to DNA Repair nicks 76  THERMOSTABLE DNA LIGASES Thermostable DNA ligases can perform ligation of duplex molecules and repair of single stranded nicks at temperatures ranging from 45 to 80°C, they provide a higher specificity and are very well suited for applications that need high stringency ligations. Thermostable DNA ligases are isolated from diverse sources such as Thermus thermophilus, Bacillus stearothermophilus, Thermus scotoductus and Rhodothermus marinus Thermostable DNA ligases are usually not a substitute for T4 or E. Coli DNA ligases but are used for very specific techniques such as Ligase Chain Reaction (LCR). LCR is a technique used to detect single base mutations 77  Enzymes that modify the ends of DNA molecules The enzymes alkaline phosphatase, polynucleotide kinase, and terminal transferase act on the termini of DNA molecules and provide important functions that are used in a variety of ways. The phosphatase and kinase enzymes, are involved in the removal or addition of phosphate groups. Bacterial alkaline phosphatase (there is also a similar enzyme, calf intestinal alkaline phosphatase) removes phosphate groups from the 5 ends of DNA, leaving a 5-OH group. 78 Enzymes that modify the ends of DNA molecules The Polynucleotide kinase is used to prevent unwanted ligation of DNA molecules, which can be a problem in certain cloning procedures. It is also used prior to the addition of radioactive phosphate to the 5 ends. Terminal transferase (terminal deoxynucleotidyl transferase) repeatedly adds nucleotides to any available 3 terminus. Although it works best on protruding 3 ends, conditions can be adjusted so that blunt-ended or 3-recessed molecules may be utilised. The enzyme is mainly used to add homopolymer tails to DNA molecules prior to the construction of recombinants. 79  Terminal Deoxynucleotidyl Transferase Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase which is capable of catalyzing the elongation of a DNA strand by the addition of nucleotides from the surrounding solution. TdT catalyses the addition of nucleotides to the 3' terminus of a DNA molecule. The preferred substrate of this enzyme is a 3'-overhang, but it can also add nucleotides to blunt ends. Cobalt is a necessary cofactor, however the enzyme catalyzes reaction upon Mg and Mn administration 80 in vitro  Terminal Deoxynucleotidyl Transferase The enzyme has a molecular weight of 32000 and consists of two subunits each with a MW of 26500 and 8000 Enzyme is also known as DNA nucleotidyltransferase (DNTT) or terminal transferase, In mammals it is encoded by DNTT gene 81  DNA with exposed 3′ OH groups, such as arise from pretreatment with phage λ exonuclease or restriction with an enzyme such as PstI. Enzyme also can add to the shielded 3′ OH of 5′ cohesive termini generated by EcoRI Typically, 10–40 homopolymeric residues are added to each end. 82  Source Calf thymus Uses To add homopolymer tails to DNA fragments. By homopolymer tailing technique, sticky ends can be built up on blunt-ended molecules. One preparation of DNA treated with enzyme terminal transferase with dATP resulting in addition of poly (dA) chain to 3’ end of each strand, other preparation adds poly (T) using TTP. Advantage is that ligation will not occur between fragments from same preparation Used for 3’end labeling of DNA fragments Addition of single nucleotide to 3’ end of DNA for in vitro mutagenesis 83 DNA Library Gene sequence are arranged in genome in a random fashion and selecting or isolating a gene is a big task especially when the genomic sequences are not known. A small portion of genome is transcribed to give mRNA where as a major portion remained untranscribed. Hence, there are two ways to represent a genomic sequence information into the multiple small fragments in the form of a library: Genomic library cDNA library Construction of Genomic Library A genomic library represents complete genome in multiple clones containing small DNA fragments. Depending upon organism and size of genome, this library is either prepared in a bacterial vector or in yeast artificial chromosome (YAC).  Steps for construction Cloning in Generation suitable Isolation of of suitable vector Transformation genomic size DNA system in suitable host DNA fragment (depending on size) Genomic DNA isolation. (A) Different steps in genomic DNA isolation. (B) Agarose gel analysis of isolated genomic DNA Construction of Genomic library Carrying capacity of different vectors Construction of cDNA Library A cDNA library represents mRNA population present at a particular stage in a organism into multiple clones containing small DNA fragments Steps in construction of cDNA library Isolation of mRNA A typical mRNA has a CAP structure at 5’, coding sequence and a poly A tail at its 3’ region Exploiting this feature, mRNA population can be isolated from RNA pool using a poly-T affinity column Steps of Isolation of mRNA Mixing of poly-T Release of total RNA containing beads with the either by a lysis buffer total RNA preparation. containing detergent or by Due to mutual exclusive homogenization in case of affinity, mRNA binds to the hard tissue poly-T beads Elute the mRNA from Wash the beads with beads; washing buffer to remove its purity can be checked non-specific cross on polyacrylamide gel contaminating species Preparation of complementary DNA First strand synthesis with reverse transcriptase Removal of RNA template Second strand synthesis Mainly two methods: Homopolymer tailing Gubber-Hoffman method Homopolymer method An oligo dT primer is used with mRNA as a template to prepare the first strand of DNA with the help of reverse transcriptase and dNTPs After the synthesis of the first strand, terminal transferase is used to add C nucleotides on 3’ of both mRNA and the newly synthesized first strand of DNA cDNA Homopolymer method DNA: RNA hybrid is loaded on an alkaline sucrose gradient. This step will hydrolyze RNA and allow the full recovery of cDNA An oligo dG primer is used with cDNA as a template to prepare the second strand of DNA with the help of reverse transcriptase and dNTPs Gubber-Hoffman method The first strand synthesis using oligo dT primer in the presence of reverse transcriptase and dNTPs DNA:RNA hybrid is treated with RNase H to produce nicks at multiple sites Gubber-Hoffman method Then DNA polymerase is used to perform DNA synthesis using multiple fragments of RNA as a primer to synthesize new DNA strands This method produces blunt end duplex DNA products Identification and Isolation of a gene There are 3 different searchable criteria to identify a particular gene from an organism DNA sequence (DNA Hybridization): This property can be used to search both genomic library and cDNA library to identify the gene Expression of a particular protein with immunogenic epitope: This property can be partially useful to screen genomic library due to truncation of a full gene or no expression of a gene fragment. But this approach suits well to screen cDNA clones Enzymatic activity: This property exploits a protein fragment’s ability to exhibit enzymatic activity. It is useful for the screening of the cDNA library but not much for the genomic library Screening by DNA Hybridization A particular DNA sequence can be identified by a complementary single stranded DNA sequence. The DNA sequence used for this purpose is called as “Probe” The position of probe can be identified by a suitable detection system The position of probe is the actual site of desirable clone of containing specific sequence. Screening cDNA library HRT and HART: cDNA library screening Hybrid Release Translation (HRT) Hybrid release translation is a means of identifying recombinant DNA clones by their ability to hybridize to, and thus promote the translation of, a specific messenger RNA in a cell-free system. This technique is used to identify the translation product encoded by a cloned gene. It depends on the ability of purified mRNA to direct synthesis of proteins in a cell free translation systems  Procedure Denaturation of cDNA. Immobilization of the denatured cDNA on nitrocellulose or nylon membrane. Incubate with the mRNA sample. The specific mRNA counterpart of the cDNA hybridizes and remains attached to the membrane.  Procedure Discard (wash) the unbound molecules. The hybridized mRNA is recovered and translated in a cell-free system. The mRNA is added to the cell-free translation system (containing ribosomes, tRNAs, and all the other molecules needed for protein synthesis; usually prepared from germinating wheat seeds or from rabbit reticulocyte). Procedure A mixture of 20 amino-acids, one labelled with S³⁵ methionine is added to the mixture. The mRNA molecules are translated into a mixture of radioactive proteins. The translated mRNA product is separated by electrophoresis and visualized via autoradiograph Hybrid Arrest Translation (HART) Hybrid arrested translation is a means of identifying recombinant DNA clones by their ability to hybridize to, and thus prevent the translation of, a specific messenger RNA in a cell-free system. Procedure: Denaturation of c-DNA. Immobilization of the denatured cDNA on nitrocellulose or nylon membrane. Incubate with the mRNA sample. The specific mRNA counterpart of the cDNA hybridizes and remains attached to the membrane. The unbound mRNAs are NOT discarded. Hybrid Arrest Translation (HART) Procedure: Denaturation of c-DNA. Immobilization of the denatured cDNA on nitrocellulose or nylon membrane. Incubate with the mRNA sample. The specific mRNA counterpart of the cDNA hybridizes and remains attached to the membrane. The unbound mRNAs are NOT discarded.  The entire sample (along with the cDNA-mRNA hybrid and the unbound mRNA) is translated in the cell-free system (containing ribosomes, tRNAs and all the other molecules needed for protein synthesis; usually prepared from germinating wheat seeds or rabbit reticulocytes). A mixture of 20 amino acids, one labelled with S³⁵ methionine is added to the mixture. The hybrid mRNA cannot direct translation, so all the proteins, except one coded by the cloned gene are synthesized. The translated mRNAs are separated by gel electrophoresis and are visualized via autoradiography. The cloned gene’s product is identified as the protein that is ABSENT from the autoradiograph. Chromosome walking Chromosome walking and chromosome jumping are two technical tools used in molecular biology for locating genes on the chromosomes and physical mapping of the genomes. Chromosome walking is a tool which explores the unknown sequence regions of chromosomes by using overlapping restriction fragments. In chromosome walking, a part of a known gene is used as a probe and continued with characterizing the full length of the chromosome to be mapped or sequenced. In chromosome walking, the ends of each overlapping fragments are used for hybridization to identify the next sequence ▪Isolation of a DNA fragment which contains the known gene or marker near target gene ▪Preparation of the restriction map of the selected fragment and sub-cloning the end region of the fragment to use as a probe ▪Hybridization of the probe with the next overlapping fragment ▪Preparation of the restriction map of the fragment 1 and sub-cloning of the end region of the fragment 1 to use as a probe for the identification of the next overlapping fragment. ▪Hybridization of the probe with the next overlapping fragment 2 ▪Preparation of the restriction map of fragment 2 and sub-cloning of the end region of the fragment 2 to serve as a probe for the identification of the next overlapping fragment Chromosome Jumping Chromosomal jumping is a technique used in molecular biology for physical mapping of genomes of the organisms. This technique was introduced to overcome a barrier of the chromosomal walking which arose upon finding the repetitive DNA regions during the cloning process. Chromosome jumping tool starts with the cutting of a specific DNA with special restriction endonucleases and ligation of the fragments into circularized loops. Then a primer designed from a known sequence is used to sequence the circularized loops. This primer enables jumping and sequencing in an alternative manner. Hence, it can bypass the repetitive DNA sequences and rapidly walk through the chromosome for the search of the target gene. Procedure Partial restriction digestion produces large overlapping DNA fragments. Fragments are circularized with DNA ligase, bringing ends that were some distance apart together. Resulting circles are cut to release the junction regions, which are cloned to form a jumping library. An end-piece cleaved from the first junction fragment is used as a probe to screen the jumping library. A second junction fragment that overlaps with the first is thus isolated. This results in the completion of the second jump. The process is repeated until the gene of interest is reached Thus the technique can be used to map the junctions in the chromosome and ‘jump’ towards the gene of interest. Thank You!