Technical & Learning Resources

Technical


Techniques
Radioimmunoassay (RIA)
Enzyme Linked Immunosorbent Assay(ELISA)
Polymerase Chain Reaction (PCR)
  Molecular biology has been revolutionized by the PCR, a method that efficiently increases the number of DNA molecules in a logarithmic and controlled fashion. The concept of DNA amplification by PCR is simple and its impact has been extraordinary. Kary Mullis conceived the PCR in 1983 and the first PCR publication appeared in 1985. Every year thereafter, the number of papers on PCR has risen exponentially.
The chemistry involved of in PCR depends on the complementarity (matching) of the nucleotide bases in the double-stranded DNA helix. When a molecule of DNA is sufficiently heated, the hydrogen bonds holding together the double helix are disrupted and the molecule separates or denatures into single strands. If the DNA solution is allowed to cool, then the complementary base pairs can reform to restore the original double helix. In order to use PCR, the exact sequence of nucleotides that flank (lay on either side of) the area of interest (the target area that needs to be amplified), must be known. This is the absolute minimum data necessary before a typical PCR reaction can be used. The researcher has to determine or use previous data (known sequence data) to synthesize complementary 5-3’ oligonucleotides (primers) of about 20 nucleotides (mer). This is referred to as 20mer primer. These serve as leaders or initiators of the replication step. The key to the reaction is that it is driven by a heat-stable polymerase molecule that reads a template DNA in the 3’-5’ direction and synthesizes a new complementary template in the 5’-3’ direction, using free dideoxy nucleoside triphosphates (dNTP’s = nucleotide bases) as building blocks.
So three major stages are ionvolved in PCR.

- Denaturation. When a double stranded DNA (dsDNA) molecule is heated to 94°C, the paired strands will separate (denature). This allows the primers access to the single stranded DNA (ssDNA) templates.
- Annealing. The reaction mixture is cooled (about 50 °C) to allow primers to select and bind (hybridize) to their complementary positions on the ssDNA template molecules.
- Elongation. The ssDNA/primer solution is heated to 72 °C. In the presence of the heat stable polymerase, PCR buffer, dNTP’s and magnesium (Mg2+) molecules, the replication procedure begins. With each repetition of this cycle, the target is doubled and soon, after about 30 cycles, the reaction will yield in excess of 1 million copies of the target DNA fragment.

There are many variations on PCR nowadays and a high degree of sophistication.

PCR has become fundamental to most areas of biological sciences and has been supported by the IAEA from 1995, where the technology was transferred successfully to African laboratories. Efforts have been sustained ever since and variations on the PCR are now used routinely in many MS laboratories.
DNA microsatellite analysis
Quantification of tannins in tree and shrub foliage
Estimation of microbial protein supply in ruminants
Technologies
Urea Molasses Multinutrient Blocks
Artificial insemination (AI) of cattle
Selective Breeding and Gene Technologies
General Information
Nuclear Glossary [106 kb, pdf]

Learning


Multimedia
DNA Replication [Quicktime]
DNA Structure [Quicktime]
Polymerase Chain Reaction [Quicktime]
Reverse Transcription Polymerase Chain Reaction (RT-PCR) [Flash Movie]
Restriction Enzymes (RE) [Flash Movie]