Week 4 (analysing cells,molecules and systems) Flashcards
What is a popular sequence aligning program for finding similar sequences?
BLAST
It scans the database for similar sequences by sliding the submitted sequence along the archived sequence until a cluster of residues falls into full or partial alignment
What have we used in recent years to to infer protein function and structure from amino acid sequences?
- NMR spectroscopy
- x-ray Crystallography
- computerisations using our own knowledge of the physical forces acting on the atoms
Why can the biochemical activity of a protein be predicted by searching databases for previously characterised proteins?
Proteins with similar structures have similar functions
How can large quantities of gene product (RNA or protein be made)?
- introducing the gene into bacteria or animal cells and coaxing these cells to over express the foreign gene
Or - synthesising the gene product in vitro
How can the biological role of any gene be assessed?
By observing the results of modifying it
What is recombinant DNA technology?
The ability to manipulate DNA with precision in a test tube or an organism and allows us to routinely study cells and their macromolecules
What manipulations are central to recombinant DNA technology
- Cleavage of DNA at specific sites by restriction nucleases, which greatly facilitates the isolation and manipulation of individual pieces of a genome
- DNA ligation, which makes it possible to seamlessly join together DNA molecules from widely different sources
- DNA cloning (through the use of cloning vectors or the polymerase chain reaction) in which a portion of the genome (often an individual gene) is “purified” away from the remainder of the genome by releatedly copying it to generate billions of identical molecules
- Nucleic acid hybridisation, which makes it possible to identify any specific sequence of DNA or RNA with great accuracy and sensitivity based on its ability to selectively bind a complementary nucleic acid sequence
- DNA synthesis, which makes it possible to chemically synthesize DNA molecules with any sequences of nucleotides, whether or not the sequence occurs in nature
- Rapid determination of the sequence of nucleotides of any DNA of RNA molecule
Explain the process of gel electrophoresis in DNA molecules?
- Each nucleotide in a nucleic acid molecule already carries a single negative charge (on the phosphate group), there is no need to add the negatively charged detergent SDS that is required to make protein molecules move uniformly toward the positive electrode
- Larger DNA fragments will migrate more slowly because their progress is impeded to a greater extent by the gel matrix
- Over several hours the DNA fragments become spread out across the gel according to size, forming a ladder of discrete bands, each composed of DNA molecules of identical length
- To separate DNA molecules longer than 500 nucleotide pairs, the gel is made of a diluted solution of agarose gel
- For DNA fragments less than 500 nucleotides long polyamide gel is used
What is the variation of agarose gel electrophoresis that makes it possible to separate extremely long DNA molecules?
Pulsed field gel electrophoresis
The DNA bands on agarose or polyacrylamide gels are invisible unless the DNA is labelled or stained by what dye?
Ethidium bromide
Which fluoresces under UV light when it is bound to DNA
What is a more sensitive dectection method following gel electrophoresis?
DNA labelled with the radioisotope 32P can be detected by placing the gel next to a piece of photographic film. The 32P atoms emit beta particles which expose the film, producing a visible record of every band on the gel
What is labelled DNA useful for?
For visualising DNA molecules in whole cells
What is hybridisation (DNA reformation)?
The reformation of hydrogen bonds between complementary base pairs
What is a DNA probe?
A short single stranded DNA molecule that is complimentary to the nucleotide sequence of interest
What is PCR used for?
Allows DNA from a specified region to be greatly amplified
