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Tools of RDT
Enzymes: Restriction Enzymes, other enzymes Vectors Host cells
Why RE?
EcoRI
Constructing rDNA!
RFLP
Vectors
Serves as a vehicle to carry a foreign DNA sequence into a given host cell. Essential features of vectors: * origin of replication (ori) * selectable marker. E.g. antibiotic resistance such as ampicillin resistance or enzymes such as beta galactosidase. * polylinker or multiple cloning site (MCS). This provides flexibility in the choice of restriction enzyme(s) that can be used for cloning. * relatively small in size
Plasmids
Extra chromosomal, self replicating, circular, double stranded DNA molecules. Found naturally in many bacteria and also in some yeast. Plasmids are not essential for the normal cell growth and division, they confer some traits on the host organism that can be a selective advantage under certain conditions. They may be present in 1 or 2 copies to multiple copies. These have been modified to carry a target gene to the host in RDT. They are the most widely used vectors.
Plasmids
One of the earliest plasmid vectors- pBR322. this contains two antibiotic resistance genes. One popular series of plasmid vectors is pUC family. They contain lacZ gene that codes for the enzyme Beta galactosidase. This region also contains a MCS and thus insertions in this region causes this gene to lose its function. This forms the direct basis of selection of recombinants. Both these plasmids replicate in E. coli only. Shuttle Vectors: vectors which can replicate in both eukaryotic cell and E. coli. These vectors contain two types of ori site and selectible markers that can function in both type of cells. E.g. Yep. In case of plants, a naturally occurring plasmid of the bacterium Agrobacterium tumefaciens called Ti plasmid has been suitably modified to act as vector.
pBR322
pUC19
Plasmids
Some times the goal of cloning is to express the cloned gene. This can be done by inserting the signals necessary for the initiation and termination of transcription and also for translation initiation into the vector adjacent to the cloning site. The vectors which contain these signals are called expression vectors.
Lytic cycle
Bacteriophage Vector
The ability to transfer DNA to specific bacterial host is used for specially designing vectors. Lambda and M13 are the most extensively used as vectors.
Lambda Bacteriophage
Double stranded, linear DNA genome of 48, 514 bp in which 12 bases at each end are unpaired but complementary. These ends are therefore sticky or cohesive and are called cos site or cohesive end site. These ends are important for packaging DNA into phage heads. The central region of its genome is not essential for the lytic cycle in E. coli and so this region can be replaced by foreign DNA. The cloning DNA size can be upto 23kb.
M13 Bacteriophage
Filamentous phage which infects E. coli having F pili. Its genome is a single stranded, circular DNA of 6407 bp. Foreign DNA can be inserted into it without disrupting any of its essential genes. As the M13 phage DNA enters the bacterial cell, it converts to a double stranded molecule known as replicative form (RF). This replicates until there are 100 copies in the cell. At this point DNA replication becomes asymmetric and ss copies of the genome are created and released from the cell as M13 particles. Advantage: genome size less than 10kb in size and RF can be purified and manipulated like a plasmid. And the genes are obtained in ss form which is useful for various techniques like DNA sequencing and site directed mutagenesis.
Cosmids
Have been constructed by combining certain features of plasmid and the cos site of lambda phage. The simplest cosmid vector contains a plasmid origin of replication, a selectable marker, suitable restriction enzyme sites and lambda cos site. The cosmids can be used to clone DNA fragments upto 45kb in length.
YAC Vectors
Yeast artificial chromosome. Used to clone DNA fragments of more than 1Mb in size. Exploited in mapping large genomes- HGP. These vectors contain telomeric sequence, centromere and an autonomously replicating sequence from yeast chromosome. Suitable restriction site and marker gene.
YAC
BAC Vectors
Bacterial Artificial Chromosome Vectors based on the natural, extra-chromosomal plasmid of E.coli- the F factor. Contains gene for replication and maintenance of the F factor, a selectable marker gene and cloning sites. They can take upto 300-350kb of foreign DNA They are used in genome sequencing projects. See table 2
Host Cells
The kind of cell depends on the aim of the cloning experiment. Gram negative bacteria E. coli is most extensively used in RDT as it is easy to handle and grow, can accept a range of vectors. Their doubling time is also short (20 minutes). For eukaryotic proteins, eukaryotic cells may be preferred. Why cant we use prokaryotic cell to express eukaryotic proteins? Yeasts have been used extensively for functional for expression of eukaryotic genes. They offer several advantages like they are simplest single celled eukaryotic organisms, genetically well characterized, easy to grow and manipulate. Plant and animal cells can also be used as host in gene manipulation and for protein expression either tissue culture or whole organism is used
DNA Library
Identification of Recombinants
Antibiotic Resistance Insertional Inactivation Blue- white selection: insertional inactivation of lac Z gene which codes for beta galactosidase. This enzyme cleaves a colourless, synthetic substrate X-Gal into a blue coloured product. Thus cells carrying rDNA will form white colonies. The above mentioned techniques are used especially in E.coli. There are several methods to detect the rDNA like digesting the plasmids obtained from the positive clones with the same restriction enzyme, by PCR and hybridization or sequencing.
Blue-white selection
PCR
Polymerase Chain Reaction. Invented by Kery Mullis in 1985. Selective amplification of a specific region of DNA. Basic principle is that when dsDNA molecule is heated to a high temperature, the two strands separate giving rise to ssDNA molecules. These single stranded molecules can be copied by DNA polymerase and thus we can generate multiple copies of the original DNA.
Requirements of PCR
DNA template to be amplified. Primers: oligo-nucleotides usually 10-18 nucleotides long. Bind to the each template strand. Two primers are required: forward primer and reverse primer. They are oriented with their ends facing each. The template region in between is amplified. DNA polymerase: it should be stable at high temperatures ~92oC. The polymerase that is generally used is Taq polymerase. This enzyme is derived from Thermus aquaticus. Deoxynucleotides, dATP, dGTP, dCTP, dTTP
PCR
Applications of PCR
Detection of pathogens. Detection of genetic basis of disease like mutations. Used in generating abundant amount DNA for DNA fingerprinting. Detecting specific microorganisms from soil samples, sediments and water. These assays offer a great sensitivity.
DNA Probes
The probe is a relatively short sequence of DNA that recognizes and binds to its complementary sequence. The binding of the DNA probes is highly specific with its complementary nucleotide sequence. Thus it is used in hybridization experiments to detect the specific nucleotide sequence. The probe is labeled to make its detection easier. We can stain a gel with ethidium bromide, but this cannot detect small amounts of DNA. The sensitivity is higher in case of probes. The probes are labeled by radioactive isotopes of P, S or a fluorescent molecule.
Hybridization Techniques
Probes are designed to detect the presence and also determine the amounts of complementary sequences in complex mixtures like cellular DNA or RNA. Southern Hybridization: originally described by Edward Southern in 1975 and has been named Southern Blotting.
Southern Hybridization
DNA Sequencing
The most fundamental method of analyzing structure of DNA is determining its sequence of bases. The first complete nucleotide sequence to be carried was for alanine-tRNA residues of yeast. It was done by Robert Holleys group at Cornell University in 1965. Two methods used for DNA sequencing developed in 1977 are: Sanger Method Maxam Gilbert Method
DNA Sequencing
Dideooxy nucleotide
Site-Directed Mutagenesis
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