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BIOTECHNOLOGY

 

M.Tevfik DORAK

 

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An overview of Biotechnology

 

Tools used in biotechnology

 

DNA extraction: Depending on the cell characteristics, DNA extraction from animal cells differs from DNA extraction from plant or prokaryotic cells. Links to Roche Product Manuals, Qiagen Handbooks. For RNA-related information, see Ambion website.

Hybridization techniques: Southern blotting, Northern blotting and in situ hybridization (including fluorescent in situ hybridisation - FISH). Hybridization techniques allows picking out the gene of interest from the mixture of DNA/RNA sequences. Hybridization only occurs between single stranded and complementary nucleic acids. The level of similarity between the probe and target determines the hybridization temperature. See an animation of Southern blotting, and an example of DNA fingerprinting.

Enzymatic modification of DNA: DNA ligase and restriction enzymes (may create sticky ends or blunt ends) are used to manipulate DNA. Most restriction enzymes recognize palindromic sequences. These are short sequences which are the same on both strands when read 5' to 3' (such as he MspI restriction site CCGG and that of EcoRI GAATTC). See the action of EcoRI. (These enzymes are called restriction endonucleases or restriction enzymes because they restrict viral replication as first discovered in 1962 by Werner Arber and only recognize site-specific or restricted sequences of DNA).

Cloning into a vector: vectors can be a plasmid (pBR322, pUC including Blue Script), lambda (l) bacteriophage, cosmid, PAC, BAC, YAC, expression vectors. The Ti plasmid is the most popular vector in agricultural biotechnology. Plasmids can accommodate up to 10 kb foreign DNA, phages up to 25 kb, cosmids up to 44 kb, YACs usually several hundred kb but up to 1.5 Mb. Gene cloning contributed to the following areas: identification of specific genes, genome mapping, production of recombinant proteins, and the creation of genetically modified organisms.

Gene libraries: Genomic (restriction digestion, sonication) or cDNA libraries are made to identify a gene. See the construction of a human genomic library.

Polymerase Chain Reaction (PCR)

Using the thermostable DNA polymerase obtained from Thermophilus aquaticus (briefly Taq), the PCR amplifies a desired sequences millions-fold. It requires a primer pair (18-30 nucleotides) to get the DNA polymerase started, the four nucleotides (dNTPs), a template DNA and certain chemicals including magnesium chloride (as a cofactor for Taq polymerase). The three steps in a cycle of the PCR - denaturation (the separation of the strands at 95o C), annealing (annealing of the primer to the template at 40 - 60o C), and elongation (the synthesis of new strands) - take less than two minutes. Taq polymerase extends primers at a rate of 2 - 4 kb/min at 72o C (the optimum temperature for its activity). Each cycle consisting of these three steps is repeated 20 - 40 times to get enough of the amplified segment. Annealing temperature of each primer is calculated using its base composition. For primers less than 20 base-long: Tm = 4(G+C) + 2(A+T). For more accurate calculation of the Tm value, visit IDT SciTools: OligoAnalyzer.

The conventional PCR is able to amplify DNA sequences up to 3 kb but the newer enzymes allow amplification of DNA fragments up to 30 kb-long. Nanogram levels of template DNA (even from a single cell) is enough to obtain amplification. The more recent 'real-time PCR' techniques are able to detect the sequence of interest in 20 picogram of total RNA. Taq polymerase has a relatively high misincorporation rate. It has been genetically modified to reduce the misincorporation rate.

See a book chapter on PCR (Strachan & Read), an article on PCR, an animation of PCR, a technical guide to PCR, and Optimization and Troubleshooting in PCR (CSH Protocols).

Different versions of PCR: Nested PCR (for increased sensitivity and specificity), reverse transcriptase (RT) PCR (starts with mRNA instead of genomic DNA), amplified fragment length polymorphism (AFLP) (replaced Southern blotting) , overlap PCR (joins two PCR products together), inverse PCR (amplifies an unknown DNA sequence flanking a region of known sequence).

Applications of PCR

1. Diagnostic use in medical genetics, medical microbiology and molecular medicine

2. HLA typing in transplantation

3. Analysis of DNA in archival material

4. Forensic analysis (DNA fingerprinting)

5. Preparation of nucleic acid probes

6. Clone screening and mapping

7. Real-time PCR and gene quantitation

DNA sequencing

The new technology allows direct sequencing of DNA fragments rather than trying to figure out the gene order, DNA mutations and new genes by traditional methods such as RFLP analysis, chromosomal walking or even transduction and conjugation experiments in bacteria. DNA sequencing has now reached the automated stage and is routinely used in many laboratories even for HLA typing. In automated sequencing, a single sequencing reaction is carried out in which the four ddNTPs are labeled with differently colored dyes. At the end of the reaction, the mixture is run in a polyacrylamide gel and the colored chains are detected as they migrate through the gel. The detection system identifies the terminal base from the wavelength of the fluorescence emitted upon excitation by a laser. The DNA polymerase used in a sequencing reaction is usually part of the E.coli polymerase known as the Klenow fragment or a genetically modified DNA polymerase from the phage T7 (Sequenase). The usual Taq DNA polymerase can also be used for this purpose.

See an animation of DNA sequencing (by dideoxy chain termination method).

 

Applications of biotechnology

1. Recombinant protein and enzyme synthesis (biopharmaceuticals)

2. Genetic modification of bacteria, animal and plant cells (genetic engineering)

3. Transgenic and knock-out animals to study gene function

4. Cloning

5. DNA fingerprinting (forensic science)

6. Biological warfare

 

Recombinant DNA in Kimball's Biology

A Virtual Tour in Agricultural Biotechnology

Genentech: The first biotechnology company

Cold Spring Harbor Protocols

 

M.Tevfik Dorak, M.D., Ph.D.

 

Last updated on 18 November 2012

 

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