SUBJECT

PCR Techniques in Plant Molecular Biology I

lecture

master

Faculty of Science

Biology MSc

2

Semesters 1-4

Autumn/Spring semester

1. Basic knowledge of the PCR method. DNA structure and DNA replication. Components of the reaction mixture. How a PCR programme is organised. Discussing each step of the PCR reaction in details. Quantification of the amplified DNA.

2. Specificity of the PCR reaction. Why does mutation occurs during the reaction? What has to be considered when oligonucleotides as primers are designed? How to calculate the melting temperature? Inhibitors and DNA contamination. Using PCR for analytical purposes. Identification of DNA sequences. Detection of the presence of a target DNA sequence in the genome. Measuring gene expression. Characterisation of the polymerase enzymes used in PCR reaction. Relation between the specificity of the reaction and the reaction mixture components.

3. Hot start PCR. Why and how to use this technique? Discussing all the advantages and disadvantages. A review of the so called Reverse transcription (RT)-PCR. Characterisation of the process and all the enzymes used for this reaction. Different strategies of the RT-PCR. Discussing all the crucial steps during the process. How to design primers for RT-PCR reaction.

4. Detection of the product of PCR reaction. Gel electrophoresis (agarose and capillary), hybridization, PCR-ELISA, real-time. Introduction to the quantitative PCR (QPCR). Kinetics of the PCR reaction. Discussion of the different approaches used for determining the quantity of the target molecule in the reaction volume (end-point, competitive and real-time PCR). Characterisation of the advantages and disadvantages of each method. Characterisation of the Rapid competitive PCR method. How to use inner standard for comparison of the quantity of the gene of interest. How to improve the detection of amplified DNA.

5. Real-time PCR (QRTPCR) How to detect the amount of amplified DNA? What has to be considered when primers and probes are designed. What are the advantages applying real-time PCR for quantity determination in simplex and multiplex reactions, discussing all the crucial points during the process.

6. Using PCR method for preparative purposes. Amplification of special sequences. “Traditional”, or standard PCR to detect the presence or absence of a special sequence in the reaction tube. What to this method can be applied for? Identification of mutant and wild type genes, or alleles. Single Nucleotide Polymorphism (SNP) as a tool for distinguishing different mutants and wild alleles from each other. Characterisation of the allele specific PCR method.

7. Advantages of the so called nested PCR. Characterisation of the method. What to be considered during primer design, like melting temperature and efficiency of the chain reaction. Multiplex PCR as a tool for detection several genes in a single tube reaction. Microsatellite analyses to characterise and analyse the plant genome. GM plant analysis by PCR method Touch down PCR as a research tool for identification of the target sequence.

8. Isolation a DNA sequence for cloning and characterisation of it. LA-PCR (Long Accurate) for amplification of large fragments. Characterisation of the polymerase enzymes purchased from biotech companies regardeing the advantages and disadvantages of each enzyme.

How to use Assymetric PCR, how to design the appropriate programme for proper amplification. Inverse PCR as a research tool for cloning and characterisation of unknown sequences

9. Degenearte PCR for gene coding for protein with known amino acid sequence. Aplication of the Thermal Asymmetric Interlaced (TAIL-PCR) method. Primer design and organising the programme for successful amplification. Using PCR for creating mutation in a target sequence. Site directed mutagenesis. Extending a known nucleotide sequence with appropriate restriction sites for special cloning. Crucila points of the primer and programme design for quick amplification.

10. Discussing the methods, including primer design and programme organisation used for site directed mutagenesis to alter the amino acid sequence of a protein (changing amino acid in a special position, deletion, insertion). Characterisation of megaprimer method. Using RNA for site directed mutagenesis without cloning the chosen gene.

11. How to apply PCR method to fuse two DNA fragments without any stuffer regions using either cloned genes or RNA. Gene shuffling. Characterisation of the Degenerate Oligonucleotide Gene Shuffling (DOGS) method in details, like primer and PCR programme design. Introduction into the method of random mutagenesis

12. Using PCR for DNA fingerprinting in plants. Review on the methods used for fingerprinting (RAPD, DAF, AP-PCR) on both theoretical and practical sides, including primer design, advantages and disadvantages of each method.

13. How to used the method of the Amplified Fragment Length Polymorphism (AFLP) as a tool for fingerprinting in plants. Microsatellite PCR for developing specific markers for genotype identification.

14. Characterisation of the methods of DNA and RNA purification from plant and comparison of different methods to each other concerning advantages and disadvantages. How to purify DNA from processed plant products. Detection of transgenic plants using different PCR methods. Searching for GM contamination in food products applying both qualitative nad quantitative PCR methods.

• Weising, Nybom, Wolff Kahl et al.: DNA Fingerprinting in Plants, CRC Press, 2005

• Review articles in English