Mol bio MOd 3

• DNA

  Deoxyribonucleic acid
• RNA


  Ribonucleic acid
• Southern blot

  Technique to detect specific DNA sequences in a DNA sample
• Dot/Slot blot

  Technique to detect specific DNA or RNA sequences by directly applying the sample to a membrane
• Solution hybridization

  Technique to measure mRNA expression when there are low amounts of target RNA
• ISH/FISH

  In-situ hybridization, technique to detect specific nucleic acid sequences in intact cells
• Northern blot

  Technique to detect specific RNA sequences in an RNA sample
• Hybridization Techniques

  • Southern blot
  • Dot/Slot blot
  • Solution hybridization
  • ISH/FISH
  • Northern blot
• Steps in Southern blot

  • DNA is isolated and cut with restriction enzymes
  • The fragments are separated by gel electrophoresis and undergo procedures of depurination and denaturation
  • The said fragments are then transferred to a solid support such as nitrocellulose
  • The DNA fragments are exposed to a labelled probe (complementary DNA or RNA) that is specific in sequence to the region of interest
• Transfer Methods for Southern Blotting

  • Capillary transfer
  • Electrophoretic transfer
  • Vacuum transfer
• Probes


  Single-stranded fragment of nucleic acid used to identify one or more sequences of interest within a large amount of nucleic acid
• DNA Probes

  • Fragment of the gene to be analyzed can be cloned on a bacterial plasmid and then isolated by restriction enzyme digestion and gel purification
  • The length of the probe will determine the specificity of the hybridization reaction. Probe lengths range from tens to thousands of base pairs
• Other Nucleic Acid Probe Types

  • Peptide nucleic acid probes
  • Locked nucleic acid probes
• Probe labeling methods

  • End-labeling
  • Nick translation
  • Random priming
• For denaturation of RNA, gel electrophoresis must be carried out under denaturing conditions for accurate transcript size assessment
• This step sets aside Northern blot from Southern blot because denaturation is carried out during electrophoresis and does not require a separate denaturation step
• Dot/Slot blots

  The target DNA or RNA is deposited directly on the membrane. Various devices, some with vacuum systems, have been designed to deposit the target on the membrane. A pipet can be used for procedures testing only a few samples. In addition, although up to several hundred test samples can be analyzed simultaneously; those samples can be tested for only one gene or gene product.
• Solution hybridization

  Has been used to measure mRNA expression when there are low amounts of target RNA. In this type of setting, both the target nucleic acid and the probe are free to interact in a reaction mixture, resulting in increased sensitivity compared to that of solid support hybridization. It also requires a smaller amount of sample.
• In-Situ Hybridization

  The target nucleic acid is found in intact cells. For probes to reach the nucleic acid, they must be small enough (≤500 bases) to penetrate the cells in question.
• HYBRIDIZATION TECHNOLOGIES


  Procedures performed that aimed at specific targets in genomic DNA (only what is needed).
• Requires visualization or detection of a specific gene or region of DNA in the background of all other genes.

  HYBRIDIZATION TECHNOLOGIES
• Southern blot

  molecular analysis of specific DNA sites within a complex background
• Hybridization

  If you take out only a specific target of DNA
• Probing of DNA

  WE apply labels on hybridized DNA
• Probed DNA

  Sequence will be used for succeeding procedures
• Southern Blot

  Designed for DNA
• Northern Blot

  Used for RNA hybridization
• Western Blot

  used for proteins
• GMOs

  Genetically Modified Organisms
• Sometimes plants and crops are manipulated to resist degradation. Sometimes we use bacteria, extract its DNA, isolate it, and clone the genes and these genes would enable resistance to insects, pests, and degradation.
• Restriction Enzyme Mapping

  Process of determining where in the DNA sequence a particular restriction enzyme recognition site is located
• Restriction site mapping

  to determine which specific genes we will be cutting, it is also to determine the sequence of genes
• Restriction site mapping

  Initially developed using small circular bacterial plasmids
• Purpose of the results of mapping

  Used to identify and characterize natural occurring plasmids and to engineer the construction of recombinant plasmids
• Number of PstI sites

  three
• PstI restriction enzyme 


  It can yield 4 restriction patterns, but it cannot indicate the order of the 4 restriction product in the original fragment
• BamHI


  It can determine the order of DNA fragments however it can only cut the DNA into 2 fragments
• RFLPs

  Restricting Fragment Length Polymorphisms
• RFLPs

  Resulting differences in the size or number of restricition fragments
• RFLPs

  The basis of first molecule-based human identification and mapping methods