Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a biochemistry and molecular biology technique[1] for isolating and exponentially amplifying a fragment or sequence of interest of DNA, via enzymatic replication, without using a living organism (such as E. coli or yeast). As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations.Invented in 1983 by Kary Mullis, PCR is now a common technique used in medical and biological research labs for a variety of tasks, such as the sequencing of genes and the diagnosis of hereditary diseases, the identification of genetic fingerprints (used in forensics and paternity testing), the detection and diagnosis of infectious diseases, and the creation of transgenic organisms. Mullis, who won the Nobel Prize for his work on PCR, credits the psychedelic drug LSD for his invention of the technique.PCR
RT-PCR 1
RT-PCR 2
Southern blot
A Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization. The method is named after its inventor, the British biologist Edwin Southern.[1] Other blotting methods (i.e., western blot, northern blot, southwestern blot) that employ similar principles, but using RNA or protein, have later been named in reference to Southern’s name. As the technique was eponymously named, Southern blot should be capitalised, whereas northern and western blots should not.
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.
- If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel may be treated with an acid, such as dilute HCl, which depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.
- If alkaline transfer methods are used, 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 provides for improved binding of the negatively charged DNA to a positively charged membrane, separates it into single DNA strands for later hybridization to the probe (see below), and destroys any residual RNA that may still be present in the DNA.
- A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top (or below, depending on the direction of the transfer) of the gel. Pressure is applied evenly to the gel (either using suction, or 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 on to the membrane; ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane.
- The membrane is then baked, i.e., exposed to high temperature (60 to 100 °C) (in the case of nitrocellulose) or exposed to ultraviolet radiation (nylon) to permanently and covalently crosslink the DNA to the membrane.
- The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA.
- 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, or by development of color on the membrane if a chromogenic detection method is used.
Northern blot
The northern blot is a technique used in molecular biology research to study gene expression. It takes its name from the similarity of the procedure to the Southern blot procedure, named for biologist Edwin Southern, used to study DNA, with the key difference that, in the northern blot, RNA, rather than DNA, is the substance being analyzed by electrophoresis and detection with a hybridization probe. This technique was developed in 1977 by James Alwine, Kemp, and George Stark at Stanford University.[1]
The gels may be run on either agarose or denaturing polyacrylamide gels depending on the size of the RNA to be detected. A notable difference in the procedure in case of agarose gels, (as compared with the Southern blot) is the addition of formaldehyde which acts as a denaturant. For smaller fragments denaturing polyacrylamide urea gels are employed.
As in the Southern blot, the hybridization probe may be made from DNA or RNA.
A variant of the procedure known as the reverse northern blot was occasionally (although, infrequently) used. In this procedure, the substrate nucleic acid (that is affixed to the membrane) is a collection of isolated DNA fragments, and the probe is RNA extracted from a tissue and radioactively labelled.
The use of DNA microarrays that have come into widespread use in the late 1990s and early 2000s is more akin to the reverse procedure, in that they involve the use of isolated DNA fragments affixed to a substrate, and hybridization with a probe made from cellular RNA. Thus the reverse procedure, though originally uncommon, enabled the one-at-a-time study of gene expression using northern analysis to evolve into gene expression profiling, in which many (possibly all) of the genes in an organism may have their expression monitored.
