recombinant DNA technology Flashcards
how does recombinant DNA technology work
it works by taking DNA from two different sources and combining that DNA into a single molecule
when this artificially created DNA is produced this is known as DNA cloning
concepts of recombinant DNA
recombinant DNA is a molecule that contains dna from two sources. also known as gene cloning .
it creates a new combination of genetic material
- human gene for insulin was placed in bacteria. the bacteria as recombinant organisms and produce insulin in large quantitys for diabetics.
genetically modified organisms are possible because of the universal nature of genetic code.
recombinant technology begins with the isolation of a gene of interest ( target gene)
the target gene is then inserted into the plasmid or vector to form a replicon
the replicon is then introduced into host cells and either expresses the protein or nor
the cloned replicon is referred to as recombinant DNA
how recombinant technology works
the steps include isolating of the target gene and the vector.
specific cutting of DNA at defined sites
joining or splicing of DNA fragments
transforming of replicon to host cell
cloning
screening of the positive cells
and either expressing or not at the end
6 basic steps to recombinant DNA experiments : step 1
isolation and purification of DNA
both vector and target dna molecules can be prepared by a variety of routine methods which are not discussed here. In some cases the target DNA is synthesised in vitro .
step 2 of recombinant DNA experiments
cleavage of DNA at particular sequences
cleaving DNA to generate fragments of defined length or with specific endpoints is crucial to recombinant DNA technology .
the DNA fragment of interest is called insert DNA
in the laboratory, DNA is usually cleaved by treating it with commercially produced nucleases and restriction endonucleases.
step 3 of recombination DNA experiments
ligation of DNA fragments
a recombination DNA molecule is usually formed by cleaving the DNA of interest to yield insert DNA and then ligating the insert DNA to vector DNA
DNA fragments are typically joined using DNA ligase
step 4 of recombination DNA experiments
introduction of recombinant DNA into compatible host cells
in order to be propagated , the recombinant DNA molecule must be introduced into a compatible host cell where it can replicate .
the direct uptake of foreign DNA by a host cell is called genetic transformation
recombinant DNA can also be packaged into virus particles and transferred to host cells by transfection.
step 5 of recombination DNA experiments
replication and expression of recombinant DNA in host cells
cloning vectors allow insert DNA to be replicated and in some cases expressed in a host cell
the ability to clone and express DNA efficiently depends on the choice of appropriate vectors and hosts
step 6 of recombination DNA experiments
identification of host cells that contain recombinant DNA of interest
vectors usually contain easily scored genetic makers or genes that allow the selection of host cells that have taken up foreign DNA.
the identification of a particular DNA fragment usually involves an additional step- screening a large number of recombinant DNA clones. this is almost always the most difficult step.
how to get the target genes
genomic DNA
artificial synthesis
PCR- amplification
RT-PCR
polymerase chain reaction
a technique called the polymerase chain reaction has revolutionised recombinant DNA technology.
it can amplify DNA from as little material as a single cell and from very old tissue such as that isolated from Egyptian mummies
RT-PCR
reverse transcription polymerase chain reaction is a variant of PCR
in RT-PCR , an RNA strand is first reverse transcribed into its DNA complement
using the enzyme reverse transcriptase , and the resulting cDNA is amplified using traditional PCR.
Vectors - cloning vehicles
cloning vectors can be plasmids , bacteriophages , viruses or even small artificial chromosomes .
most vectors contain sequences that allow them to be replicated autonomously within a compatible host cell , whereas minority carry sequences that facilitate integration into the host genome.
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 called a polylinker
Types of vector
Plasmid Vectors
Bacteriophage Vectors
Virus vectors
Shuttle Vectors- can replicate in either prokaryotic or eukaryotic cells.
Yeast Artificial Chromosomes as Vectors
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.
Plasmids can replicate autonomously within a host,
and they frequently carry genes conferring resistance to antibiotics such as tetracycline, ampicillin, or kanamycin.
The expression of these marker genes can be used to distinguish between host cells that carry the vectors and those that do not
pBR322
pBR322 was one of the first versatile plasmid vectors developed; it is the ancestor of many of the common plasmid vectors used in biochemistry laboratories.
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: confer resistance to ampicillin, and confer resistance to tetracycline.
pBR322 contains a number of unique restriction sites that are useful for constructing recombinant DNA.
Restriction Enzymes Cut DNA Chains at Specific Locations
Restriction enzymes are endonucleases produced by bacteria that typically recognize specific 4 to 8 bp sequences, called restriction sites, and then cleave both DNA strands at this site.
Restriction sites commonly are short palindromic sequences; that is, the restriction-site sequence is the same on each DNA strand when read in the 5′ → 3′ direction.
form sticky ends: single stranded ends that have a tendency to join with each other ( the key to recombinant DNA)
Blunt ends or sticky ends
Each enzyme recognizes and cleaves a specific double-stranded DNA sequence that is 4–7 bp long.
These DNA cuts result in blunt ends (eg, Hpa I) or overlapping (sticky) ends (eg, BamH I) , depending on the mechanism used by the enzyme.
Restriction enzymes
Restriction enzymes are named after the bacterium from which they are isolated
For example, Eco RI is from Escherichia coli, and Bam HI is from Bacillus amyloliquefaciens .
The first three letters in the restriction enzyme name consist of the first letter of the genus (E) and the first two letters of the species (co).
These may be followed by a strain designation (R) and a roman numeral (I) to indicate the order of discovery (eg, EcoRI, EcoRII).
Identification of Host Cells Containing Recombinant DNA
Once a cloning vector and insert DNA have been joined in vitro, the recombinant DNA molecule can be introduced into a host cell, most often a bacterial cell such as E. coli.
In general, transformation is not a very efficient way of getting DNA into a cell because only a very small percentage of cells take up recombinant DNA.
Consequently, those cells that have been successfully transformed must be distinguished from the vast majority of untransformed cells.
Identification of host cells containing recombinant DNA requires genetic selection or screening or both.
In a selection, cells are grown under conditions in which only transformed cells can survive; all the other cells die.
In contrast, in a screen, transformed cells have to be individually tested for the presence of the desired recombinant DNA.
Normally, a number of colonies of cells are first selected and then screened for colonies carrying the desired insert.
Screening (Strategies)
Gel Electrophoresis Allows Separation of Vector DNA from Cloned Fragments
Cloned DNA Molecules Are Sequenced Rapidly by the Dideoxy Chain-Termination Method
The Polymerase Chain Reaction Amplifies a Specific DNA Sequence from a Complex Mixture
Blotting Techniques Permit Detection of Specific DNA Fragments and mRNAs with DNA Probes
Types of blotting techniques
Southern blotting
Southern blotting techniques is the first nucleic acid blotting procedure developed in 1975 by Southern.
Southern blotting is the techniques for the specific identification of DNA molecules.
Northern blotting
Northern blotting is the techniques for the specific identification of RNA molecules.
Western blotting
Western blotting involves the identification of proteins.
Antigen + antibody
Applications of Recombinant DNA Technology
Analysis of Gene Structure and Expression
Pharmaceutical Products
Drugs
Vaccines
Genetically modified organisms (GMO)
Transgenic plants
Transgenic animal
Application in medicine
