11-10-2012, 03:18 PM
Introduction to Cloning and Recombinant DNA Technology
Cloning and Recombinant.ppt (Size: 6.58 MB / Downloads: 316)
Deoxyribonucleic acid (DNA) is a long double-stranded chain of nucleotides
DNA is the hereditary material passed on from generation to generation.
DNA is made up of four nucleotides: A, C, G, and T.
A always pairs with T.
C always pairs with G.
The two strands of DNA are in an antiparallel configuration.
Two complementary DNA strands will separate when heated, and will spontaneously pair together again (hybridize) when cooled.
What Does It Mean: “To Clone”?
Clone: a collection of molecules or cells, all identical to an original molecule or cell
To "clone a gene" is to make many copies of it - for example, by replicating it in a culture of bacteria.
Cloned gene can be a normal copy of a gene (= “wild type”).
Cloned gene can be an altered version of a gene (= “mutant”).
Recombinant DNA technology makes manipulating genes possible.
Restriction Enzymes
Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes”.
Cut up “foreign” DNA that invades the cell.
Type II and III restriction enzymes cleave DNA chains at selected sites.
Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage.
An enzyme that recognizes a 6-base sequence is called a "six-base cutter”.
Plasmids – vehicles for cloning
Plasmids are naturally occurring extrachromosomal DNA molecules.
Plasmids are circular, double-stranded DNA.
Plasmids are the means by which antibiotic resistance is often transferred from one bacteria to another.
Plasmids can be cleaved by restriction enzymes, leaving sticky or blunt ends.
Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid.
Cloning Vectors
A cloning vector is a plasmid that can be modified to carry new genes.
Plasmids useful as cloning vectors must have:
An origin of replication.
A selectable marker (antibiotic resistance gene, such as ampr and tetr).
Multiple cloning site (MCS) (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers).
Easy to purify away from host DNA.
Chimeric Plasmids
Named for mythological beast (chimera) with body parts from several creatures.
After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted.
Ends of the plasmid/fragment are closed to form a "recombinant plasmid”.
Plasmid can replicate when placed in a suitable bacterial host.
PCR and prenatal diagnosis
For prenatal diagnosis, PCR used to amplify DNA from fetal cells obtained from amniotic fluid.
Single base changes then detected by one or more of following:
-dot blot (spot hybridization) with oligonucleotides specific for known mutation.
-restriction enzyme analysis (RFLP).
-direct sequencing of DNA.
Important to be certain of result so combination of two methods provides confirmation.
Many other conditions can be detected with same approach, including:
-Tay-Sachs disease, phenylketonurea, cystic fibrosis, hemophilia, Huntingdon's disease, Duchenne muscular dystrophy (DMD).
PCR to detect HIV
PCR allows the direct detection of HIV genomes in patient blood before the appearance of HIV antibodies.
viral DNA/RNA only represents a minute proportion of total cell DNA.
Only a small fraction of blood cells are infected (1/10,000).
also require high degree of specificity while targeting conserved regions of DNA to guard against high level of genetic variability characteristic of retroviruses.
High risk of cross-contaminating sample with small amounts of amplified DNA from previous sample requires extra precautions to prevent false-positives.
PCR can detect 10-20 copies of viral DNA from 150,000 human cells.
PCR can be more rapid and accurate than other diagnostic tests
Diagnosis of the middle ear infection known as otitis media. The technique has detected bacterial DNA in children's middle ear fluid, signaling an active infection even when culture methods failed to detect it.
Lyme disease, the painful joint inflammation caused by bacteria transmitted by tick bites, can be diagnosed by detecting the disease organism's DNA contained in joint fluid.
PCR is the most sensitive and specific test for Helicobacter pylori, the disease organism now known to cause almost all stomach ulcers.
PCR can detect three different sexually transmitted disease organisms on a single swab (herpes, papillomaviruses, and chlamydia).
PCR in Forensics
Crucial forensic evidence may be present in very small quantities.
often too little material for direct DNA analysis.
but PCR can generate sufficient DNA from a single cell.
PCR also possible on extensively degraded DNA.
examples include DNA from single dried blood spot, saliva (on cigarette butt), semen, tissue from under fingernails, hair roots.
Other advantages of PCR in forensic science are:
relatively simple to perform and simple to standardize.
results obtainable within 24 hours.
The major legal problems with PCR are the potential for cross-contamination between samples and the complexity of explaining what the results mean to the jury.