Cloning Vectors Gene Cloning Cloning a definition From
- Slides: 33
Cloning Vectors
Gene Cloning
Cloning - a definition § From the Greek - klon, a twig § An aggregate of the asexually produced progeny of an individual; a group of replicas of all or part of a macromolecule (such as DNA or an antibody) § An individual grown from a single somatic cell of its parent & genetically identical to it § Clone: a collection of molecules or cells, all identical to an original molecule or cell
DNA CLONING A method for identifying and purifying a particular DNA fragment (clone) of interest from a complex mixture of DNA fragments, and then producing large numbers of the fragment (clone) of interest.
Gene cloning § When DNA is extracted from an organism, all its genes are obtained § In gene (DNA) cloning a particular gene is copied (cloned)
Why Clone DNA? § A particular gene can be isolated and its nucleotide sequence determined § Control sequences of DNA can be identified & analyzed § Protein/enzyme/RNA function can be investigated § Mutations can be identified, e. g. gene defects related to specific diseases § Organisms can be ‘engineered’ for specific purposes, e. g. insulin production, insect resistance, etc.
Sources of DNA for Cloning § 1) Chromosomal DNA § 2) RNA converted to c. DNA § 3) PCR-amplified DNA
PCR-amplified DNA
Cloning Tools § § § Restriction endonucleases Ligase Vectors Host Methods for introducing DNA into a host cell
Cutting DNA § Restriction endonucleases (restriction enzymes) • sticky ends • blunt ends § Nomenclature • • • Eco. RI E = genus (Escherichia) co = species (coli) R = strain I = # of enzyme
Blunt & Sticky ends
Pasting DNA § Complementary ends (sticky ends) H-bond § Ligase forms phosphodiester bond to seal strands together.
Vectors
Cloning vectors Allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level. 1 Plasmid vectors 2 Bacteriophage vectors 3 Cosmids 4 BACs & YACs
Plasmid vectors are double-stranded, circular, selfreplicating, extra-chromosomal DNA molecules. § Advantages: • Small, easy to handle • Straightforward selection strategies • Useful for cloning small DNA fragments (< 10 kbp) § Disadvantages: • Less useful for cloning large DNA fragments (> 10 kbp)
Plasmid vectors § Plasmids are circular DNA molecules present in the cytoplasm of the bacteria § Capable of autonomous replication § Can transfer genes from one cell to other § Act as vectors in genetic engineering. § Can also present in Yeasts
Plasmid vectors § may encode genetic information for properties 1 Resitance to Antibiotics 2 Bacteriocins production 3 Enterotoxin production 4 Enhanced pathogen city 5 Reduced Sensitivity to mutagens 6 Degrade complex organic molecules T. V. Rao MD
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.
Plasmid vectors
Bacteriophage vectors § Advantages: • Useful for cloning large DNA fragments (10 - 23 kbp) • Inherent size selection for large inserts § Disadvantages: • Less easy to handle
l vectors § Left arm: • head & tail proteins § Right arm: • DNA synthesis • regulation • host lysis § Deleted central region: • integration & excision • regulation
Bacteriophage
Cosmid vectors Combine the properties of plasmid vectors with the useful properties of the l cos site § Advantages: • Useful for cloning very large DNA fragments (32 - 47 kbp) • Inherent size selection for large inserts • Handle like plasmids § Disadvantages: • Not easy to handle very large plasmids • (~ 50 kbp)
§ A cosmid is a type of hybrid plasmid that contains a Lambda phage cos sequence. Cosmids' (cos sites + plasmid = cosmids) DNA sequences are originally from the lambda phage. [1] 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. [2] § Cosmids can contain 37 to 52 (normally 45) kb of DNA, limits based on the normal bacteriophage packaging size. They can replicate as plasmids if they have a suitable origin of replication: for example SV 40 ori in mammalian cells, Col. E 1 ori for double-stranded DNA replication or f 1 ori for single-stranded DNA replication in prokaryotes. 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. [3]
§ Unlike plasmids, they can also be packaged in phage capsids, which allows the 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.
§ Cos sequences are ~200 base pairs long and essential for packaging. They contain a cos. N site where DNA is nicked at each strand, 12 bp apart, by terminase. This causes linearization of the circular cosmid with two "cohesive" or "sticky ends" of 12 bp. (The DNA must be linear to fit into a phage head. ) The cos. B site holds the terminase while it is nicking and separating the strands. The cos. Q site of next cosmid (as rolling circle replication often results in linear concatemers) is held by the terminase after the previous cosmid has been packaged, to prevent degradation by cellular DNases.
l ZAP
BACs and YACs BACs : Bacterial Artificial Chromosomes YACs : Yeast Artificial Chromosomes § Advantages: • Useful for cloning extremely large DNA fragments (100 - 2, 000 kbp) • This is very important for genome sequencing projects § Disadvantages: • Not easy to handle extremely large DNA molecules
BAC vector § ori. S and ori. E mediate replication § par. A and par. B maintain single copy number § Chloramphenicol. R marker
YAC vector large inserts ARS URA 3 telomere centromere replication origin HIS 3 markers telomere § Capable of carrying inserts of 200 - 2000 kbp in yeast
What determines the choice vector? § insert size ¡ vector size ¡ restriction sites ¡ copy number ¡ cloning efficiency ¡ ability to screen for inserts ¡ what down-stream experiments do you plan?
- Cloning vector
- Cosmid vector slideshare
- Gene cloning
- Function of plasmid
- Plasmid
- Genez molecular clones
- Gene by gene test results
- Chapter 17 gene expression from gene to protein
- Pros of cloning
- Gateway technology with clonase ii
- Reproductive vs therapeutic cloning
- Suspension cloning
- Identity cloning and concealment
- Cloning in nature
- Cloning and sequencing explorer series
- Genetic engineering steps
- Inverse pcr
- Clock gate cloning
- Positional cloning
- Pixpsd
- Advantages and disadvantages of cloning
- Advantages and disadvantages of cloning
- Advantages of cloning
- Reproductive cloning
- Human cloning cons
- Gsm cloning
- Process of cloning
- What is cloning
- Metyláza kofaktor
- Therpeutic cloning
- Agauti
- What is gene pool
- Img srcx onerroralert1
- Example of vectors