Molecular Biology; 2

DNA Continued

Hybridisation-based techniques;

- In situ DNA hybridisation; Presence and localisation/organisation of DNA sequence (gene/genome)

DNA probes: stretches of single-stranded DNA used to detect the presence of complementary nucleic acid sequences (target sequences) by hybridization. DNA probes are labelled, for example with radioisotopes, epitopes, biotin or fluorophores to enable their detection

FISH (flouresent in-situ hybridisation) used to locate dna section within a chromosome.

Fluorophores are characterised by their excitation and emission spectra

-In situ RNA hybridisation; Presence and localisation of m-RNA sequences (levels of gene expression/transcriptome)

 Electophoresis- separates the molecules in a mixture by causing them to migrate under the influence of an electric field (polinucleotides are negatively charged, based on the phosphate groups, nucleotides chain move to the positively charged pole) Smaller fragments move faster, thus travel further.

DNA digestion; We need enzymes that hydrolize the phosphodiester bonds in the DNA backbone leading to dissociation of the adjacent nucleotides.

Two types:

Endonucleases: DNAses that cleave DNA within a polynucleotide chain.

Exonucleases: DNases that cleave only nucleotides at the ends…