* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download enzymes and vectors
Comparative genomic hybridization wikipedia , lookup
Gene expression wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Molecular evolution wikipedia , lookup
List of types of proteins wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Biosynthesis wikipedia , lookup
Non-coding DNA wikipedia , lookup
Community fingerprinting wikipedia , lookup
DNA vaccination wikipedia , lookup
DNA supercoil wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Molecular cloning wikipedia , lookup
RESTRICTION ENZYMES • Restriction Enzymes scan the DNA code • Find a very specific set of nucleotides • Make a specific cut RESTRICTION ENZYMES • Restriction enzymes, also called restriction endonucleases, recognize, bind to specific sequences in double-stranded DNA, and cleave the DNA. • They are usually isolated from bacteria. • The role of these enzymes in bacteria is to "restrict" the invasion of foreign DNA by cutting it into pieces. • Hence, these enzymes are known as restriction enzymes. • The cell's own DNA is not degraded, because the sites recognized by its own restriction enzymes are methylated. • Many restriction enzymes have been purified and characterized. • The names of restriction enzymes consist of a three-italic-letter abbreviation for the host organism. • For example, restriction enzyme EcoRⅠis from Escherichia coli. • The first three letters in the name of the enzyme consist of the first letter of the genus (E) and the first two letters of the species (co), which are followed by a strain designation (R) and a roman numeral (Ⅰ)to indicate the order of discovery. NOMENCLATURE OF RESTRICTION ENZYME • Each enzyme is named after the bacterium from which it was isolated using a naming system based on bacterial genus, species and strain. For e.g EcoRI Derivation of the EcoRI name Abbreviation Meaning Description E Escherichia genus co coli species R RY13 strain I First identified order of identification in the bacterium Restriction enzyme nomenclature Why the funny names? • EcoRI – • BamHI – • DpnI – • HindIII – • BglII – • PstI – • Sau3AI – • KpnI – Escherichia coli strain R, 1st enzyme Bacillus amyloliquefaciens strain H, 1st enzyme Diplococcus pneumoniae, 1st enzyme Haemophilus influenzae, strain D, 3rd enzyme Bacillus globigii, 2nd enzyme Providencia stuartii 164, 1st enzyme Staphylococcus aureus strain 3A, 1st enzyme Klebsiella pneumoniae, 1st enzyme • There are three types of restriction enzymes, designatedⅠ,Ⅱ,andIII. • TypesⅠand Ⅲ contain the activities of both the endonuclease and methylase. • TypeⅠ restriction enzymes cleave DNA at random sites. • Type Ⅲ restriction enzymes cleave the DNA about 25 bp from the recognition sequence. • Both types of enzymes require ATP for energy supply. • TypeⅡ restriction enzymes, require no ATP, and usually cleave the DNA within the recognition sequence itself. • So typeⅡ restriction enzymes have extraordinary utility in DNA recombination. • Many type Ⅱ restriction enzymes recognize specific sequences of 4 to 6 base pairs and cleave a phosphodiester bond in each strand in this region. • One unique feature of restriction enzymes is that the nucleotide sequences they recognize are palindromic, or inverted repeats. • It cuts one strand of the DNA double helix at one point and the second strand at a different, complementary point. • For example, the sequence recognized by a restriction enzyme EcoRⅠ is GAATTC. • In each strand, the enzyme cleaves the GA phosphodiester bond on the 5' side of the symmetric axis. • The arrow indicates the cleavage site. •If the cleavage site is not at the center, the restriction enzyme ( e.g., EcoRⅠ) will generate cohesive ends(sticky ends), which can base-pair with other DNA fragments cleaved by the same restriction enzyme. • If the cleavage site is at the center, the restriction enzyme (e.g., HpaⅠ) will generate blunt ends blunt end sticky end The specificities of several of these enzymes are shown in Table 6-2. Table 6-2 Commonly used restriction enzymes Some more examples of restriction sites of restriction enzymes with their cut sites: HindIII: 5’ AAGCTT 3’ 3’ TTCGAA 5’ BamHI: 5’ GGATCC 3’ 3’ CCTAGG 5’ AluI: 5’ AGCT 3’ 3’ TCGA 5’ HaeIII HaeIII is a restriction enzyme that searches the DNA molecule until it finds this sequence of four nitrogen bases. 5’ TGACGGGTTCGAGGCCAG 3’ 3’ ACTGCCCAAGGTCCGGTC 5’ 5’ TGACGGGTTCGAGGCCAG 3’ 3’ ACTGCCCAAGGTCCGGTC 5’ Once the recognition site was found HaeIII could go to work cutting (cleaving) the DNA 5’ TGACGGGTTCGAGGCCAG 3’ 3’ ACTGCCCAAGGTCCGGTC 5’ • This enzyme is used to covalently link or ligate fragments of DNA together • Isolated from viruses • Also occurs in E.coli and eukaryotic cells • It also participates in DNA repair process • DNA ligase catalyses the formation of phosphodiester bond between the 5’-phosphate of one strand of DNA or RNA and the 3’-hydroxyl of another. • The DNA ligase used in molecular cloning differ in their abilities to ligate substrate,such as blunt ended duplex DNA:RNA hybrid or ssDNAs. MECHANISM OF DNA LIGASE • The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor"). ATP is required for the ligase reaction, which proceeds in three steps: (1) Adenylation (addition of AMP) of a residue in the active center of the enzyme, pyrophosphate is released. (2) Transfer of the AMP to the 5' phosphate so-called donor, formation of a pyrophosphate bond; (3) Formation of a phosphodiester bond between the 5' phosphate of the donor and the 3' hydroxyl of the acceptor. • Depending up on the source,the enzyme requires either ATP or NAD+ as cofactors Bacteriophage T4 DNA Ligase (ATP) • The most widely used DNA ligase is derived from the T4 bacteriophage. • It is a monomeric polypeptide • MW 68KDa is encoded by bacteriophage gene30. • It has broder specificity and repairs single stranded Nicks in duplex DNA, RNA or DNA:RNA hybrids. APPLICATION 1. Ligation of cohesive ends 2. Ligation of blunt ended termini 3. Ligation of synthetic linkers or adapter E.Coli DNA ligase • It is derived from E.coli cell and requires NAD+ as cofacter. • It is a monomeric enzyme of MW 74KDa which catalyzes the formation of the phosphodiester bond in duplex DNA containing cohesive ends. • This enzyme has narrower substrate specificity, making it a useful tool in specific application. APPLICATION 1) Ligation of cohesive ends 2) Cloning of full length cDNA E.coli DNA ligase has been employed in a procedure for high efficiency cloning of full length cDNA Taq DNA ligase [NAD+ ] • The gene encoding thermostable ligases have been identified from several thermophilic bacteria. • Several of this ligase have been cloned and expressed to high levels in E.coli • Retain their activities after exposure to higher temp for multiple rounds. • It is uses in DNA amplificaton reaction to detect mutation in mammalian DNA. T4 RNA Ligase • T4 RNA ligase is the only phage RNA ligase that used in genetic engineering. • This enzyme catalyzed the phosphodiester bond formation of RNA molecule with hydrolysis of ATP to PPI • It is monomeric enzyme a product of the T4 gene 63 APPLICATION 1) Production of elongated molecules 2) Modification of internal nucleotide 3) Stimulation of T4 DNA ligase activity ALKALINE PHOSPHATASE • Alkaline phosphatase(ALP), is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins and alkaloids. • The process of removing the phosphate group is called Dephosphorylation. ENZYMES USED IN MOLECULAR BIOLOGY ALKALINE PHOSPHATASE • Alkaline phosphatase removes 5' phosphate groups from DNA and RNA. • It will also remove phosphates from nucleotides and proteins. • These enzymes are most active at alkaline pH ALKALINE PHOSPHATASE • There are two primary uses for alkaline phosphatase in DNA manipulations: • Removing 5' phosphates from plasmid and bacteriophage vectors that have been cut with a restriction enzyme. In subsequent ligation reactions, this treatment prevents self-ligation of the vector and thereby greatly facilitates ligation of other DNA fragments into the vector (e.g. subcloning). • Removing 5' phosphates from fragments of DNA prior to labeling with radioactive phosphate. Polynucleotide kinase is much more effective in phosphorylating DNA if the 5' phosphate has previously been removed DEPHOSPORYLATED VECTOR R.E.S WITH COMPATIBLE ENDS POLYMERASES • Group of enzymes that catalyses the synthesis of nucleic acid molecules are collectively referred to as polymerases. • Three important polymerases are given below:• DNA –dependant DNA polymerase :- that copies DNA from DNA. • RNA dependant DNA polymerase (Reverse Transcriptase): that synthesizes DNA from RNA. • DNA dependant RNA polymerases: that produces RNA from DNA DNA POLYMERASE • A DNA polymerase is an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand. • DNA polymerases are best known for their role in DNA replication in which the polymerase" reads” an intact DNA strand as a template and uses it to synthesize the new strand. • The process copies a pieces of DNA. • DNA polymerases use a mg++ for catalytic activitiy. • Type . Pol I, pol II, pol III EXONUCLEASE • Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end of a polynucleotide chain. • A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or the 5’ends occurs. TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE • Terminal Deoxynucleotidyl Transferase, also known as TdT and terminal transferase. • TdT catalyses the addition of nucleotides to the 3’ terminus of a DNA molecule. • Cobalt is a necessary cofactor POLYNUCLEOTIDE KINASE • Polynucleotide kinase (PNK) is an enzyme that catalyzes the transfer of a phosphate from ATP to the 5’ end of either DNA or RNA. REVERSE TRANSCRIPTASE • This enzyme by using the template of RNA ,synthesize the new strand of DNA . RNA cDNA dsDNA NUCLEASES •Nucleases are a class of enzymes called hydrolases that catalyzes the hydrolysis of nucleic acids(DNA,RNA) in all organisms including plants and humans. •Nucleases are usually specific in action, ribonucleases acting only upon ribonucleic acids (RNA) and deoxyribonucleases acting only upon deoxyribonucleic acids (DNA). •There are two types of nucleases: Endonucleases and exonucleases Exonucleases degrade nucleic acids from one end of the molecule. They opearate either in 5’ 3’ or 3’ 5’ direction. Endonucleases degrade nucleic acids at specific internal sites, reducing it to smaller and smaller fragments. Restriction enzyme,a endonuclease due to it’s cleavage at specific nucleotide sequence find so much importance in recombinant DNA technlology. •Nuclease cleavage sites •(phosphodiester linkage) •Cleavage at bond A generates a 5’phosphate and a 3’ OH terminus •Cleavage at bond B generates a 3’phosphate and a 5’hydroxyl terminus ROLE OF NUCLEASES •Processes under control of nucleases are protective mechanisms against "foreign” (invading) DNA •degradation of host cell DNA after virus infections •DNA repair •DNA recombination • DNA synthesis III. Vectors for Gene Cloning INTRODUCTION • A cloning vector is a DNA molecule in which foreign DNA can be inserted or integrated and which is further capable of replicating within host cell to produce multiple clones of recombinant DNA. • Examples: Plasmids,phage or virus Characteristics It should be able to replicate autonomously. Origin of replication. Selectable markers. Restriction sites. A. Requirements of a vector to serve as a carrier molecule • The choice of a vector depends on the design of the experimental system and how the cloned gene will be screened or utilized subsequently • Most vectors contain a prokaryotic origin of replication allowing maintenance in bacterial cells. • Some vectors contain an additional eukaryotic origin of replication allowing autonomous replication in eukaryotic cells. All cloning vectors have in common at least one unique cloning site, a sequence that can be cut by a restriction endonuclease to allow site-specific insertion of foreign DNA. The most useful vectors have several restriction sites grouped together in a multiple cloning site (MCS) called a polylinker B. Main types of vectors • Plasmid, • bacteriophage, • cosmid, • bacterial artificial chromosome (BAC), • yeast artificial chromosome (YAC), • retrovirus, • baculovirus vector…… C. Choice of vector • Depends on nature of protocol or experiment • Type of host cell to accommodate rDNA • Prokaryotic • Eukaryotic PLASMID VECTORS Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cell’s chromosomal DNA. These extra chromosomal DNAs, which occur naturally in bacteria and in lower eukaryotic cells (e.g., yeast), exist in a parasitic or symbiotic relationship with their host cell. Naturally occurring bacterial plasmids size range is 5000 to 400,0000 bp. Plasmid PLASMID VECTORS Advantages: Small, easy to handle Straightforward selection strategies Useful for cloning small DNA fragments (< 10kbp) Disadvantages: Less useful for cloning large DNA fragments (> 10kbp) A plasmid vector for cloning 1. Contains an origin of replication, allowing for replication independent of host’s genome. 2. Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistance blue-white screening 3. Contains a multiple cloning site (MCS) 4. Easy to be isolated from the host cell. 4362 bp SELECTIVE MARKER • Selective marker is required for maintenance of plasmid in the cell. • Because of the presence of the selective marker the plasmid becomes useful for the cell. • Under the selective conditions, only cells that contain plasmids with selectable marker can survive • Genes that confer resistance to various antibiotics are used. • Genes that make cells resistant to ampicillin, neomycin, or chloramphenicol are used ORIGIN OF REPLICATION • Origin of replication is a DNA segment recognized by the cellular DNA-replication enzymes. • Without replication origin, DNA cannot be replicated in the cell. MULTIPLE CLONING SITE • Many cloning vectors contain a multiple cloning site or polylinker: a DNA segment with several unique sites for restriction endo- nucleases located next to each other • Restriction sites of the polylinker are not present anywhere else in the plasmid. • Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector TYPES OF PLASMIDS Conjugative:- (stringent plasmid) • Carry a set of transfer genes that facillitates bacterial conjugation • Are large, show stringent control of DNA replication and present in low numbers • Low copy number = 1-4 copies / cell Non conjugative:- (relaxed plasmid) • If they do not posses such genes. • Are small,show relaxed control of DNA replication and present in high number • High copy number = 10-100 copies / cell F plasmid : • posses genes for their own transfer from one cell to another R plasmid: • carry genes resistance to antibiotics. pBR322 pBR322 was one of the first versatile plasmid vectors developed; it is the ancestor of many of the common plasmid vectors used in laboratories. • Derived from E. coli plasmid ColE1), which is 4,362 bp DNA. • pBR322 is named after Bolivar and Rodriguez, who prepared this vector. pBR322 contains an origin of replication (ori) and a gene (rop) that helps regulate the number of copies of plasmid DNA in the cell. There are two marker genes: confers resistance to ampicillin, and confers resistance to tetracycline. pBR322 contains a number of unique restriction sites that are useful for constructing recombinant DNA. It has unique restriction sites for 20 restriction endonucleases. pBR322 1. Origin of replication 2. Selectable marker 3. unique restriction sites • Another series of plasmids that are used as cloning vectors belong to pUC series (after the place of their initial preparation I.e. University of California). • These plasmids are 2,700 bp long and possess • Ampicillin resistance gene • The origin of replication derived from pBR322 and • The lacz gene derived from E.coli. • Within the lac region also having unique restriction sites. • When DNA fragments are cloned in this region of pUC, the lac gene is inactivated. • On the other hand, pUC having no inserts are transformed into bacteria, it will have active lac Z gene and therefore will produce blue colonies, thus permitting identification of colonies having pUC vector with cloned DNA segments. pUC19 2.68kbp Bacteriophage lambda (λ) A virus that infects bacteria o BACTERIOPHAGE VECTORS • BACTERIAL VIRUS • Infects bacterial cells by injecting their genetic material (DNA or RNA) • Follow either Lytic cycle and Lysogenic cycle • Commonly used E.Coli phages are l (lambda) , M 13 ,Fd phages. • Most efficient than plasmid for cloning of large fragments of over 25 kb. • Easy to screen • MAINLY l - widely used • Larger capacity of insert than PLASMIDS Bacteriophage vectors • Advantages: • Useful for cloning large DNA fragments (10 - 23 kbp) • Inherent size selection for large inserts • Disadvantages: • Less easy to handle Bacteriophage BACTERIOPHAGE LAMBDA • Phage lambda is a bacteriophage or phage, i.e. bacterial virus, that uses E. coli as host. • Its structure is that of a typical phage: head, tail, tail fibres. • Lambda viral genome: 48.5 kb linear DNA with a 12 base ssDNA "sticky end" at both ends; these ends are complementary in sequence and can hybridize to each other (this is the cos site: cohesive ends). • Infection: lambda tail fibres adsorb to a cell surface receptor, the tail contracts, and the DNA is injected. • The DNA circularizes at the cos site, and lambda begins its life cycle in the E. coli host. Bacteriophage lambda COS site: Cohesive “sticky” ends Lysis Replication ori Lysogeny Head Tail Circularized lambda • Lambda genome is approximately 49 kb in length. • Only 30 kb is required for lytic growth. • Thus, one could clone 19 kb of “foreign” DNA. • Packaging efficiency 78%-100% of the lambda genome. BACTERIOPHAGE LAMBDA . DNA cloning using phages as vectors PHAGE M13 VECTOR COSMID VECTORS • Hybrid molecules containing components of both lambda and plasmid DNA • Lambda components: COS sequences (required for in vitro packaging into phage coats) • Plasmid DNA components: ORI + Antibiotic resistance gene • Cosmids can carry up to 50 kb of inserted DNA. • Cloning sites will be part of vector COSMID VECTORS • RDNA is packaged using extracts of coat and tail proteins derived from normal lambda components BUT cannot be packaged after introduced into host cell because rDNA does not encode the genes required for coat proteins • The cos sequences occurs at one end of lambda DNA molecules and it is responsible for its insertion into the phage capsid. • Cos sites allows them to be packaged into capsids. • After packaged it used to infect E. coli. • After infection of E. coli, rDNA molecules replicate as plasmids CLONING BY USING COSMID VECTORS SHUTTLE VECTORS • Hybrid molecules designed for use in multiple cell types • Multiple ORIs allow replication in both prokaryotic and eukaryotic host cells allowing transfer between different cell types • Examples: • E. coli yeast cells • E. coli human cell lines • Selectable markers and cloning sites SHUTTLE VECTORS • Possess two origin for replication (ori E & ori Euk). • Can be expressed in either host • Can be grown in one host and shifted to another host • ori E functions in E. coli & ori Euk functions in eukaryotic cells like yeast. YEAST VECTORS • Yeast is a unicellular eukaryotic micro organism. • Reproduce sexually as well as asexually • Contains their own plasmid (6318 bp long) • Present in high number copy Yeast plasmid contains: • Origin of replication (ori) • Cis action region (REP 3) • Two genes ( REP 1 & REP 2) Yeast artificial chromosomes (YACs) • Hybrid molecule containing components of yeast, protozoa and bacterial plasmids • Yeast: • ORI = ARS (autonomously replicating sequence) • Selectable markers on each arm (TRP1 and URA3) • Yeast centromere • Protozoa= Tetrahymena • Telomere sequences (yeast telomeres may also be used) • Bacterial plasmid • Polylinker • Can accommodate >1Mb (1000kbp = 106 bp) YAC vector large inserts URA3 HIS3 ARS telomere telomere centromere markers replication origin Capable of carrying inserts of 100 kbp in yeast Bacterial artificial chromosomes (BACs) • Based on F factor of bacteria (imp. In conjugation) • Can accommodate 300 kb of inserts • Advantage is the instability problems of YACs can be avoided • F factor components for replication and copy # control are present • Selectable markers and cloning sites available BAC vector oriS and oriE mediate replication parA and parB maintain single copy number ChloramphenicolR marker Human artificial chromosomes • Developed in 1997 – synthetic, self-replicating • ~1/10 size of normal chromosome • Microchromosome that passes to cells during mitosis • Contains: • ORI • Centromere • Telomere • Protective cap of repeating DNA sequences at ends of chromosome (protects from shortening during mitosis) • Histones provided by host cell What determines the choice vector? insert size vector size restriction sites copy number cloning efficiency ability to screen for inserts APPLICATIONS OF RDNA TECHNOLOGY IN PHARMACEUTICAL APPLICATIONS • Several proteins are created from recombinant DNA (recombinant proteins) and are used in medical applications. • Hematopoietic growth factor. • Interferon’s • Hormones • Recombinant protein vaccines • Tissue/bone growth factors and clotting factors • Biological response modifiers • Monoclonal/Diagnostic/Therapeutic antibodies • Recombinant proteins is extensively used in biotechnology, medicine and research. 100 PRODUCTS EXTRACTED FROM TISSUE/ PRIMARY CELLS Product Insulin Growth hormone Interferon Urokinase Factor VIII Extracted from.... Pancreas; bovine or porcine Human pituitary glands Viral activation of cells Human urine Pooled human blood PROBLEMS OF EXTRACTION FROM ANIMAL/ HUMAN SOURCES • Small quantities available • Non-human proteins cause immunogenicity • contamination with viruses or prions INSULIN Insulin • Hormone produced by beta cells in the pancreas → allows glucose to pass into cells → suppresses excess production of sugar in the liver and muscles → suppresses breakdown of fat for energy ANIMAL CELL PRODUCTS – RECOMBINANT PROTEINS beta cells in pancreas Preproinsulin (109 A.A) Proinsulin (86 A.A) Insulin(51 A.A) + C-peptide Computer-generated image of insulin hexamers highlighting the threefold symmetry, the zinc ion holdin it together and the histidine residues invlolved in zincbinding INSULIN 51 amino acids 5,8808 molecular weight ANIMAL CELL PRODUCTS – RECOMBINANT PROTEINS • Insulin produced from pig pancreas cells → structure of insulin differs slightly between species → the C-terminal amino acid of the B chain = alanine (threonine in humans) • two problems associated with porcine insulin → causes immunogenic response in some diabetic patients → supply of pancreas fluctuates with meat trade INSULIN SYNTHESIS • Prepared by synthetic gene or from mRNA separated from rat pancrease. • Chemically synthesized DNA sequence for A & B chains of insulin. • The synthetic A & B genes are separately inserted into two pBR 322 plasmid by the side of galactosidase gene. • The recombinant plasmids are separately transferred into E.Coli cells. INSULIN SYNTHESIS • The bacterial cells are grown in large fermenter by using proper nutrients and optimized physical conditions. • The product contains large chimeric protein consisting of the A and B chain attached to naturally occurring E.Coli protein. • These chains are detached from protein through cyanogen bromide. • Chain A and B are joined in vitro to form insulin by sulphonating the two peptides with sodium disulphonate and sodium sulphite. Producing A and B chains separately INSULIN PRODUCTION 112 THANK YOU -PHARMA STREET