RL1 - Nucleic Acids: Detection & Quantification

October 20th

How do Molecular Biology Techniques enhance our ability to detect & analyze gene products?

through 

• qualitative analysis

  • nature of molecules in question

  • size

  • nucleotide composition - the sequence

  • conformation configuration

  • structure — what are the molecules like in 3D space? 

• quantitative analysis 

  • determine levels if the gene products

    • i.e. tumor markers such as tumor supressor genes: are they over/under expressed?

    • e.g. allows us to make diagnostic conclusions

based on what property do molecular probes function

based on property of nucleic acids to base pair with their complements

What is the mechanism of using a molecular probe? — 5 steps

1. complex mixture of macromolecules

2. binding to a membrane 

• on an agarose gel w/ electrophoresis we have nucleic acid which we can then transfer to solid support (so that there for good)

3. Probe for specific target

• make a reverse complement (this is the probe) for target sequence that you label (both need to be ssDNA)

4. remove non-specific

5. Target detection

• we have size markers so the quantity is now known

How do we label single stranded oligonucleotides using polynucleotide kinases

• We use a known seq that corresponds to the gene of interest

  • We synthesize the oligonucleotide to have a reverse complement ot that seq

• The polynucleotide kinases (PNK) will phosphorylate nucleotides by transferring the gamma phosphate of ATP to the free hydroxyl at the 5’ end of the synthetic oligonucleotide

  • We can buy the radioactive phosphate

  • We use an enzyme ot cleave the gamma phosphate and attach it to the synthetic oligonucleotide (that doesn’t itself have a phosphate)