PLANT BIOTECHNOLOGY & GENETIC ENGINEERING (3 CREDIT HOURS) LECTURE 5 POLYMERASE CHAIN REACTION SOUTHERN BLOTTING NORTHERN BLOTTING WESTERN BLOTTING BULK SEGREGANT ANALYSIS

POLYMERASE CHAIN REACTION PCR is a technique in which DNA duplication is carried out under in vitro conditions, so that multiple copies of the required segment of DNA are obtained. The experiment is carried out in “eppendorf tubes” in which a polymerase enzyme, the four deoxyribonucleic phosphates of DNA, and a primer (so that the reaction is initiated) are added. When the reaction is initiated, production of copies of DNA flanked by pair of primers takes place in the PCR machine (thermal cycler). The major steps involved in PCR are the following: Double stranded DNA (ds DNA) or cDNA is denatured by heating at 95-98℃. Synthetic oligonucleotide primers with sequences complementary to target DNA are added in excess to the reaction mixture along with the nucleotides. When the reaction mixture is cooled to 37 ℃, primers bind to the complementary sequences present on the single stranded template DNA.

POLYMERASE CHAIN REACTION The thermostable enzyme “Taq polymerase” obtained from the thermo-tolerant bacterium Thermus aquaticus is added to the reaction mixture. It catalyses the extension of the primers at 72 ℃ from the end of the target region by adding appropriate nucleotides to form a complementary DNA sequence. This cycle is continuously repeated to get multiple copies of the target DNA. Theoretically PCR yields 2n copies of target DNA (n = number of cycles). Incubation for 3-4 hours (25-30 cycles) results in million copies of target DNA.

POLYMERASE CHAIN REACTION PCR, a simple and rapid procedure, is utilized to confirm whether a putatively transgenic plant that has survived selection is indeed transgenic. Usually, two primers (one forward and one reverse) specific for the selectable marker (bar gene, for example) are used in a PCR reaction with genomic DNA extracted from the transgenic plants. A thermostable DNA polymerase amplifies the region between the two primers during the multiple amplification cycles of the PCR, which yields a DNA fragment of predicted size (the length equal to the number of base pairs between the two primers in the transgene). This fragment is easily detected on an agarose gel by staining with ethidium bromide. PCR is a very sensitive and rapid method for identification of transgenic plants in the seedling stage and requires only a small amount of plant tissue.

SOUTHERN BLOTTING/ HYBRIDIZATION ANALYSIS In Southern blotting, DNA fragments from transgenic plants generated by digestion with restriction enzyme(s) are first separated according to fragment size by electrophoresis through an agarose gel. The DNA fragments then are transferred to a solid support, such as a nylon membrane or a nitrocellulose sheet. The transfer is affected by simple capillary action, sometimes assisted by suction or electric current. The DNA binds to the solid support, usually because the support has been treated to carry a net positive charge, or some other means of binding such as inducing covalent binding of the DNA to the support. DNA fragments maintain their original positions in the gel after transfer to the membrane. Hence, larger fragments will be localized toward the top of the membrane and smaller fragments toward the bottom. The positions of specific fragments can then be determined by “probing” the membrane. The probe consists of the DNA fragments of interest, such as a cloned gene, which has been labeled with a radioactive isotope or some other compound that allows its visual detection. Under the proper set of conditions, the denatured single-stranded probe will hybridize to its complementary single strands of genomic DNA affixed to the membrane.

SOUTHERN BLOTTING/ HYBRIDIZATION ANALYSIS In this way, the size of the fragment on which the probe resides in the genomic DNA can be determined. In transgenic plant experiments, the Southern blot often is used to determine whether an introduced gene is indeed present in the plant DNA and whether multiple transgenic plants carry the introduced gene on the same size of DNA fragment (suggesting independent transformation events). The results of Southern blots also indicate whether a single copy of the gene has been inserted or if multiple copies are likely to be present.

NORTHERN BLOTTING Northern blotting – the name was derived as a play on words from the Southern blot – is very similar to the Southern blot, except that instead of restriction enzyme-digested DNA, native RNA is separated according to size by electrophoresis through an agarose gel and then transferred to a solid support. The rest of the Northern blot procedure is very similar to that of the Southern blot and it is used to determine whether the introduced gene has been transcribed into messenger RNA and accumulates in the transgenic plant.

WESTERN BLOTTING The Western blotting procedure detects the protein of the transgene in an extract of proteins prepared from various parts of the transgenic plants and is, therefore, an assay for a functional transgene. In this technique the proteins are first electrophoresed in an SDS-polyacrylamide gel and the proteins are then transferred to nitrocellulose of polyvinylidene difluoride (PVDF) membrane by electrophoretic transfer. The membrane is then treated with an antibody specific for the protein encoded by the transgene followed by a second antibody coupled to an enzyme, which can act on a chromogenic (or fluorogenic) substrate leading to visualization of the transgene protein with increased sensitivity. The expression level of the protein can be quantified using known amounts of the transgenic-encoded protein.

BULK SEGREGANT ANALYSIS Monogenic qualitative traits When DNA of ten plants are bulked into one pool, all alleles must be present Two bulked pools of segregants differing for one trait, will differ only at the locus harboring that trait. Based on pooling DNA from individuals with similar phenotypes Any difference in RFLP or RAPD pattern between these two bulks would be linked to the locus upon which the bulk was developed. Analyze 4 samples (2 parents + 2 bulks) with all polymorphic markers – run the resulting marker on the whole population – single marker analysis

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