PRINCIPLES OF CROP PRODUCTION ABT-320 (3 CREDIT HOURS)

1. PRINCIPLES OF CROP PRODUCTIONABT-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

2. TECHNIQUES FOR GENETIC ENGINEERING 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.

3. ISOLATION OF TOTAL CELLULAR DNA • DNA is very easily damaged by shear forces, even rapid stirring of solution can break even high DNA molecular weight into much shorter fragments. Consequently, DNA is recovered from cells by gentlest possible method of rupture, in the presence of EDTA to chalate the Mg+2 ions needed for DNase activity. Cell walls, if present, are digested enzymatically (e.g. lysozyme treatment of bacteria) and the cell membrane is solubilised using detergent. Cell disruption is performed at 4℃ using autoclaved glassware and solutions to destroy DNase activity. • After release of nucleic acids from the cells, RNA can be removed by treatment with ribonuclease (RNase). The other major contaminant, protein, is removed by shaking the solution gently with water-saturated phenol, or with phenol/chloroform mixture, either of which will denature protein but not nucleic acids. Centrifugation of the emulsion formed by this mixture produces a lower, organic phase, separated from the upper aqueous phase by an interface denatured proteins. The aqueous solution is recovered and deproteinised repeatedly, until no matter is seen at interface. Finally, the deproteinised DNA preparation is mixed with ethanol and allowed to precipitate out in freezer. After centrifugation, the DNA pellet is dissolved in a buffer containing EDTA for protection against DNases and this solution can be stored at 4℃ for at least a month.

4. ISOLATION OF TOTAL CELLULAR DNA • DNA solutions can be stored frozen, but repeated freezing and thawing tends to damage long molecules by shearing; so preparation in frequent use are normally stored at 4℃. • This procedure is suitable for total cellular DNA. If the DNA from specific organelle or viral particle is needed, it is best to isolate the organelle or virus before extracting its DNA.

5. NUCLEIC ACID HYBRIDIZATION 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.

6. 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.

7. NUCLEIC ACID HYBRIDIZATION • When employed analytically, hybridization is normally performed using one labeled sequence, termed the probe, and an unlabelled sequence called the target. Probe is a short synthetic oligo deoxyribonucleotide which is complementary to target DNA sequence. Target DNA may be obtained from total cellular DNA preparations. The probe is labeled by incorporation of either radioactively labeled nucleotides or with some chemicals. The probe is the known, pure species in the hybridization and the target is the unknown species to be identified. • The target will most often form part of a mixture of unrelated nucleic acid sequences. The probe is usually added in considerable excess over the target, and its concentration determines the rate of reaction. Hybridization reaction time is extended to ensure complete reaction. The target and probe can be hybridized to each other in solution, or alternatively the target can be immobilized on an inert support such as nitrocellulose.

8. METHODS FOR LABELING NUCLEIC ACIDS 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.

9. METHODS OF LABELING NUCLEIC ACID & PROBES There are five basic methods for labeling nucleic acids. These are: • Nick translation • Primer extension • Methods based on RNA polymerase • End labeling methods • Direct labeling methods

10. 32P-labelling of duplex DNA by nick translation. Asterisks indicate radiolabelled phosphate groups.

11. NICK TRANSLATION • This is done by making single-strand cuts (nicks) in the double stranded DNA molecule by brief exposure to a dilute solution of an endonuclease (usually deoxyribonuclease 1 of E. coli). DNA polymerase 1 is then used in the presence of at least one radioactive precursor to “translate” the nick along the molecule in the 5’ to 3’ direction. The net result is that a nonradioactive strand of DNA is replaced by a radioactive strand. The DNA is then denatured and used as a radioactive probe in hybridization experiments (Southern blots, Northern blots etc). • Nick translation can be used with a variety of labels to generate probes suitable for most hybridization applications. It is also appropriate for the generation of biotinylated probes.

12. PRIMER EXTENSION METHOD • Primers are synthetic oligodeoxyribonucleotides which are complementary to specific regions of known vector DNA. The 3’ termini of these primers serve as initiation site for template dependent DNA synthesis by enzymes like DNA polymerase 1. • DNA polymerase works by extending a short double-stranded region made by annealing an oligonucleotide primer to the single-stranded template. Thus this method of uniform labeling requires a primer which matches the probe sequence. Radiolabelling of primers can be done with two methods. • If the probe sequence is not known then random oligonucleotide labeling can be used. It is often in the case when natural cellular DNA is used. These primers are made by adding a mixture of all four bases at each step in the chemical synthesis reaction. The DNA is denatured and the two complementary strands are copied in the presence of labelled primers as well as nucleoside triphosphates. The polymerase used is Klenow fragment derived from DNA polymerase-I of E. coli.

13. PRIMER EXTENSION METHOD • Chance homology ensures that these primers anneal to the separated DNA strands at many points along their length, thus providing a base for polymerase to initiate DNA synthesis. This is only one of several uniform labeling methods. • The second method uses a unique primer to restrict labeling to a particular sequence of interest. In the primer extension method, it is essential to use a polymerase lacking a 5’ 3’ exonuclease activity otherwise degradation of the primer will occur. The Klenow fragment of E. coli DNA polymerase I, which lacks the 5’ 3’ exonuclease activity has been used successfully. • It is an ideal method for situations where high specific activity and low probe concentrations are frequently employed.

14. 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.

15. METHODS BASED ON RNA POLYMERASES • RNA polymerases catalyzes the synthesis of RNA from nucleoside triphosphates using a DNA template. Thus they can incorporate labeled ribonucleotides into RNA during transcription if such labeled nucleotides are provided to it. If a specific site of a vector or DNA is transcribed in such way, RNA probes (or transcripts) of defined length and sequence can be obtained. • Initially RNA probes were obtained from RNA polymerase from E. coli. This enzyme when used in vitro exhibited very little template and promoter specificity and therefore produces transcripts which have been initiated and terminated at random. Today, RNA polymerases from a number of phages are used, which possess a high degree of specificity. Thus transcription can be limited to sequences cloned downstream of an appropriate promoter.

16. END-LABELLING OF NUCLEIC ACIDS • A wide variety of techniques is available for introducing label at either the 3’ or 5’ ends of linear DNA or RNA. Usually only a single label is introduced at the terminus. Nucleic acid can be 5’ end labeled using T4 polynucleotide kinase. Radiolabeled phosphate group is donated by [γ32-P] ATP to DNA or RNA containing a 5’-hydroxyl terminus. This is termed as a forward reaction. • If 5’-phosphate group is present in DNA or RNA, then it is removed with alkaline phosphatase. This reaction is driven by excess ADP which causes the enzyme to transfer the terminal 5’-phosphate from DNA to ADP. This is known as exchange reaction. The DNA is rephosphorylated by transfer of labeled γ-phosphate from [γ32-P] ATP.

17. The 5’-end labelling reaction using T4 polynucleotide kinase

18. END-LABELLING OF NUCLEIC ACIDS The major advantages of 5’-end labeling are: • Both DNA and RNA can be labeled. • Location of labeled group is known. • Very small fragments can be labeled. • Restriction digest fragment can be labeled.

19. CHOICE OF LABEL • There are two technical parameters, resolution and sensitivity, which determines the success of probe application. High degree of resolution is required to know the relative position of a nucleic acid fragment. High sensitivity is necessary because sequence of interest may be present at low abundance. • Other factors are probe stability, safety and ease of use. Broadly labels can be categorized into radioactive and nonradioactive types.

20. RADIOACTIVE LABELS 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.

21. NON-RADIOACTIVE LABELS A number of non radioactive labels for probes are available but biotin is widely used.

22. BIOTIN LABELLED PROBES 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: • assays employ non-toxic materials, with longer half-life. • can be prepared in advance in bulk and stored at -20℃ for repeated uses. • Detection of hybrids is much faster than by radioactive probes.

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