Oligonucleotide Synthesis + Blotting Flashcards
What is an oligonucleotide?
A DNA sequence made up of a ‘few’ nucleotides (3-100 nt)
Which direction does DNA get synthesized in phosphoramidite chemistry?
3’-5’
What is a probe?
A small sequence of DNA that is complementary to a specific sequence of target DNA. Can be labelled with a flourescent tag.
What are the key parameters that allow for hybridisation of DNA to work?
Sequence Concentration Affinity of probe to sequence Sequence length Ionic Strength pH Temperature Mismatches
What is the basic calculation for hybridisation melting temperature?
2 x (A+T) + 4 x (G+C)
What are the 5 types of probes?
Genomic DNA Oligonucleotides Complementary DNA Heterologous probe PCR-generated fragments
Explain nick translation labelling?
DNase 1 enzyme creates a nick in dsDNA on one strand
DNA exonuclease chews that section of DNA out
DNA pol adds oligo labelled probe at complementary sites (labelled dNTPs)
Heat and anneal, you have a labelled probe
Explain random hexamer labelling?
dsDNA is heated to form ssDNA
Add hexamer nucleotide probes that will complementary base pair to strand of DNA
Ligase enzyme stitches them together with more ntds
heat and anneal
Probe is formed
What are flourochrome probes?
Modified nucleotides using enzymes i.e biotinylated dNTP
Used for greater sensitivity
How are total proteins detected?
Stains like coomassie blue
How are specific proteins detected?
Using antibodies labelled with enzymes, flourophores, etc
What is blotting?
Transfer of molecules to a thin membrane for further analysis.
Immobalize and fix on the membrane using heat or UV light.
What is low stringency?
The conditions of the reaction are not very specific so the probe may not bind specifically to the sequence of DNA.
Blotting of DNA is ?
Blotting of RNA is ?
Blotting of proteins is ?
Southern blotting
Northern blotting
Western blotting
How does DNA move onto the nylon membrane in blotting (3 ways)?
Capillary action or by using probes or by using electric (electroblotting)
