PRINCIPLES OF CROP PRODUCTION ABT-320 (3 CREDIT HOURS). LECTURE 14 TECHNIQUES FOR GENETIC ENGINEERING, ISOLATION OF TOTAL CELLULAR DNA NUCLEIC ACID HYBRIDIZATION METHODS FOR LABELING NUCLEIC ACIDS CHOICE OF LABELS. TECHNIQUES FOR GENETIC ENGINEERING.
TECHNIQUES FOR GENETIC ENGINEERING,
ISOLATION OF TOTAL CELLULAR DNA
NUCLEIC ACID HYBRIDIZATION
METHODS FOR LABELING NUCLEIC ACIDS
CHOICE OF LABELS
Recombinant DNA technology deals with isolation and manipulation of genes. Before nucleic acids can be cut or otherwise manipulated, they must be either isolated and purified or artificially synthesized. The isolated genes are also sometimes sequenced for a better understanding of its structure that needs to be manipulated.
If a double-stranded DNA molecule is exposed to high temperature, or to very alkaline conditions, then the two strands will break apart. The molecule is said to have become denatured. The temperature at which denaturation occurs is termed as melting temperature or Tm. If the denatured DNA is returned to a temperature below its Tm or to neutral pH when alkali was used to denature it, each strand will, after a time, find its complementary strand. The two strands will ‘zipper’ back together to re-form a double stranded DNA molecule. This ability of complementary sequences to anneal, or hybridize, to one another is called nucleic acid hybridization. This technique helps in determining the gene structure and in identifying molecules which contain same sequences of nucleotides. In a complex mixture of nucleic acid molecules, nucleic acid hybridization technique helps in separation of complementary sequence.
The principle of hybridization using an inert support. The target DNA is most often localized to one part of the support and, in most cases, the aim of the hybridization experiment is to identify this region. The probe is prelabelled by some method, most often by the incorporation of radioactivity. After hybridization, the annealed probe is detected by autoradiography.
For identification of hybridized nucleic acid duplexes, labeling of probe or target is necessary. As probe is short synthetic oligonucleotide it can be easily labeled. A 32P isotopic label can be added to the 5’ hydroxyl group using polynucleotide kinase to a probe. Alternatively, labeled nucleotides can be incorporated at the 3’ end using terminal transferase. Because label is added at only one place within the oligonucleotide, such end-labelled probes contain relatively little radioactivity per unit weight of DNA. This low specific activity limits their sensitivity as probes. If a high specific activity probe is required, then some method of uniform labeling must be used. Here the probe sequence is copied along part or all of its length using DNA polymerase and labeled nucleoside triphosphate.
There are five basic methods for labeling nucleic acids. These are:
The principle of random primed (oligo-) labelling. The DNA to be used as a probe is denatured by heating and mixed with hexanucleotides of random sequence which then act as primers for DNA synthesis.
The major advantages of 5’-end labeling are:
These labels have wider applications as they can be easily detected with autoradiography. Their detection gives two important information, firstly about occurrence of hybridization between probes and target DNA and secondly about their position. Radioactive methods using 32P are easily detectable. They are used often.
A number of non radioactive labels for probes are available but biotin is widely used.
Biotinylated probes are prepared through a nick-translation reaction by replacing nucleotides with biotinylated derivatives. After hybridization and washing, detection of hybrids is done by adding avidine and going through a series of cytochemical reactions which finally give a blue color whose intensity is proportional to the amount of biotin in the hybrid. There are several advantages of using biotinylated probes. The major advantages of using biotinylated probes are:
THE END to be used as a probe is denatured by heating and mixed with hexanucleotides of random sequence which then act as primers for DNA synthesis.