prehybridization(1) (3) steps of dna extraction .pptx

prehybridization(1) (3) steps of dna extraction .pptx

• 1.
  PREHYBRIDIZATION
• 2.
  • Pre-hybridization isa process that involves incubating a membrane with a blocking reagent to prevent the probe from binding directly to the membrane, and instead to the DNA bands. It's a key step in hybridization experiments, which also include hybridization and post- hybridization washing.
• 3.
  • Purpose • Pre-hybridizationblocks free binding sites on the membrane. This prevents the probe from binding to the membrane instead of the DNA bands. • Reagents • The pre-hybridization buffer is usually the same as the hybridization buffer, but without the probe. SDS is an important component of the pre-hybridization buffer. • Procedure • The membrane is blocked by shaking it softly in the pre-hybridization solution for 30 minutes.
• 4.
  • Hybridisation • Theutility of nucleic acid hybridization is based on the original discovery by Watson and Crick that DNA is a double-stranded molecule held together by hydrogen bonds between complementary bases. Because of the complementarity of the two strands, denatured DNA derived from the same parent molecule can reanneal (or hybridize) under conditions of appropriate pH, temperature, and ionic strength. Nucleic acid hybridization is usually performed with target DNA that has been immobilized onto membranes (from Southern blots, by spot loading DNA onto membranes or after lysis of the bacterial colonies on a replica filter) and a probe in a liquid phase.
• 5.
  • Hybridization conditionscan be adjusted by changing the temperature and salt concentration. The melting temperature of DNA is the temperature at which the DNA strands separate. If the DNA of two species is very similar, the melting temperature will be higher.
• 6.
  • In Southernblotting the DNA is usually denatured with alkali, so it is bound as single strands to the membrane and ready for hybridization. The labeled probe is usually double-stranded and has to be denatured before it is added. • The blot with the DNA is hybridized to a labeled probe, washed extensively and then the hybridized probe must be detected (usually by autoradiography or immunological methods).
• 7.
  • The hybridizationand washing conditions are critical. If the probe and target are 100% identical in sequence, then a high stringency hybridization can be carried out. The stringency is determined by the hybridization temperature and the salt concentration in the hybridization buffer (high temperature and low salt is more stringent as only perfectly matched hybrids will be stable). For probes that do not match the target completely (a less specific probe), the stringency must be reduced to a level that allows imperfect hybrids to form. If the stringency of the hybridization (or washing) is too low, then the probe may bind to too many sequences to be useful.
• 8.
  • All protocolsemploy a reagent to block non-specific attachment of the probe to the membrane (i.e. to reduce so-called background hybridisation signal). This blocking reagent may contain a protein, such as casein from nonfat dried milk or bovine serum albumin, often in combination with denatured, fragmented salmon sperm DNA (or any other heterologous DNA of high complexity) and a detergent, such as SDS. Often just a very high concentration of SDS is used as a blocking agent.
• 9.
  • All protocolsemploy a reagent to block non-specific attachment of the probe to the membrane (i.e. to reduce so-called background hybridisation signal). This blocking reagent may contain a protein, such as casein from nonfat dried milk or bovine serum albumin, often in combination with denatured, fragmented salmon sperm DNA (or any other heterologous DNA of high complexity) and a detergent, such as SDS. Often just a very high concentration of SDS is used as a blocking agent.
• 10.
  • The rateof DNA reannealing is very much depending on the concentration of the probe. To reduce the hybridization time and the amount of probe required, only just enough hybridization buffer is used to cover the membrane (keeping the concentration of the probe as high as possible), with constant agitation. This is often accomplished by using special bottles which are rotated horizontally in a hybridization oven.
• 11.
  • After theincubation of the blot with the probe, washing steps are necessary to remove excess probe (sticking non-specifically to the membrane). The stringency of the washings is important for reasons mentioned above. Stringent washing is: high temperature, low salt. A commonly used washing solution is SSC (Saline Sodium Citrate, a salty sodium citrate solution, a mixture of NaCitrate and NaCl.
• 12.
  Southern and Northernblotting protocols involve the following major steps: • Purification of DNA/RNA: Extract and purify the DNA/RNA from either cells or tissue sources. • Digestion of DNA: Digest the DNA into fragments with restriction enzymes. This step is not required for RNA. • Gel electrophoresis: Separate the DNA fragments on agarose gel. The RNA samples can be separated on agarose gel with formaldehyde as the denaturing agent that limits secondary structures of RNA molecules. • Transfer: Transfer the DNA/RNA fragments from the gel onto a nylon membrane.
• 13.
  • Prehybridization (Blocking):Wash the nylon membrane with a prehybridization solution containing salmon sperm DNA to block non-specific DNA interactions and reduce background noise. Alternatively, use the PerfectHyb™ Plus buffer, which doesn't require salmon sperm DNA for blocking. • Preparation of probe: Prepare fresh probe DNA and label with 32 P alpha-labeled dCTP. • Hybridization: Incubate the blot with labeled probe. • Detection of probe: Detect the probe and the DNA/RNA sequence of interest by exposure to film at -80 °C.