lec 7 - blotting + multiplex pcr biochemistry..pptx

lec 7 - blotting + multiplex pcr biochemistry..pptx

1. Multiplex PCR

2. Advantages: • This technique has the potential to produce considerable savings in time and effort within the laboratory Disadvantages: • Optimization is difficult ; since many sets of forward and reverse primers are to be designed for use. • Increases cost . • Presence of multiple primer may lead to cross hybridization with each other and the possibility of mis -priming with other templates.

3. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes , i.e., their base pair length , should be different enough to form distinct bands when visualized by  gel electrophoresis . Alternatively, if amplicon sizes overlap, the different amplicons may be differentiated and visualised using primers that have been dyed with different colour fluorescent dyes.

4. Blotting Techniques

5. Nitrocellulose membranes are a popular matrix used in protein blotting because of their high protein-binding affinity , compatibility with a variety of detection methods ( chemiluminescence , chromogenic , and fluorescence), and the ability to immobilize proteins, glycoproteins , or nucleic acids.

6. Southern blotting

7. Method Restriction  endonucleases  are used to cut high-molecular-weight DNA strands into smaller fragments. The DNA fragments are then  electrophoresed  on an  agarose gel  to separate them by size . - The DNA gel is placed into an alkaline solution (typically containing  sodium hydroxide ) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged thymine residues of DNA to a positively charged amino groups of membrane, separating it into single DNA strands for later  hybridization  to the probe, and destroys any residual RNA that may still be present in the DNA.

8. - A sheet of  nitrocellulose  (or, alternatively,  nylon )  membrane  is placed on top of the gel. Pressure is applied evenly to the gel by placing a stack of paper towels and a weight on top of the membrane and gel, to ensure good and even contact between gel and membrane. Buffer transfer by  capillary action  from a region of high  water potential  to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel onto the membrane. the DNA binds to the membrane due to the negative charge of the DNA and positive charge of the membrane . - The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours (standard conditions; nitrocellulose or nylon membrane) to permanently attach the transferred DNA to the membrane.

9. - The membrane is then exposed to a  hybridization probe —a single DNA fragment with a specific sequence. The probe DNA is labelled so that it can be detected, usually by incorporating  radioactivity  or tagging the molecule with a  fluorescent   dye . - After hybridization, excess probe is washed from the membrane, and the pattern of hybridization is visualized on  X-ray  film by  autoradiography  in the case of a radioactive or fluorescent probe.

10. Northern Blotting

11. Western blotting or immunoblotting A technique in which proteins are separated by gel electrophoresis and transferred to a membrane sheet. A specific protein is then identified through its reaction with a labeled antibody.

12. PROCEDURE - The proteins of the sample are separated using  gel electrophoresis ( PAGE, Poly Acrylamide Gel Electrophoresis) and are then transferred onto nitro cellulose to which they bind. - Then radiolabelled specific antibody is added on such membrane and it binds only to specific complementary protein. - The antibody is labelled with iodine-125 and the signal is detected again with autoradiography. -If radio active label is not used, bound antibody may be detected by a second antibody tagged with an enzyme , which allows to visualize the protein-Ab1-Ab2 complex.