Analysis of Nucleic Acids Flashcards
What is a replicon? Example?
Something capable of independent replication. Plasmids e.g.
Describe in vivo cell cloning
DNA molecules are constructed in vitro by cutting a target DNA sequence and a replicon with the same restriction endonuclease so that the end of the two DNA are compatible. Purify and mix then join with DNA ligase. Put the rejoined DNA in host cells. Propagate colonies which have the replicon in (kill others with antibiotics). Expand cell culture, isolate and purify the recombinant DNA.
Explain the function of restriction endonuclease, how the cuts can form and how host DNA is protected
Enzymes that cleave DNA at certain recognition sequences (usually 4-8bp palindromic sequences). Can produce blunt ends where DNA is perfectly cleaved in half or sticky ends where there are overhanging bases/assymetrically. Host DNA is protected by methylation of a base in the recognition site, so only DNA from invading organisms will be killed.
Which nucleic acid does Southern blotting use?
DNA target + DNA probe
Which nucleic acid does Northern blotting use?
RNA target + DNA probe
Which nucleic acid does Colony blotting use?
Bacterial DNA target + DNA probe
Which nucleic acid does tissue in situ use?
RNA target RNA probe
Which nucleic acid does chromosome blotting use?
Chromosome target + DNA probe
Which nucleic acid do microarrays use?
Immobilised DNA or oligonucleotide probe _ target DNA solution
What does the energy needed to denature bonds depend on? (3)
Strand length, base composition and chemical environment (monovalent cations stabilise the DNA duplex by neutralising the charge of the phosphate backbone, denaturants (e.g. urea/formamide) destabilise the DNA duplex)
What 3 factors should be considered when designing primers?
Length (about 20 nucleotides to give specificity), base composition (tandem repeats can form hairpins) and the 3’ end must avoid complementarity or it may form primer dimers.
What can a microarray be used for?
Expression profiling
