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0.1 Basic principles of genetics  (Page 15/19)

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Cloning into a vector. Vectors can be a plasmid (pBR322, pUC including Blue Script), lambda (λ) 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. Link to examples of plasmids .

Lecture 15. genes can be manipulated by molecular tools ii

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 sequence 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).

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 an article on PCR , an animation of PCR , and a technical guide to PCR .

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); real-time PCR (detects the sequence of interest in very small quantity).

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.

5. Preparation of nucleic acid probes.

6. Clone screening and mapping.
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Source:  OpenStax, Genetics. OpenStax CNX. Jul 29, 2009 Download for free at http://cnx.org/content/col10782/1.1
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