Lec 17 - Advanced Molecular Techniques

this allows us to take a DNA sequence and use ddNTP (terminates elongation) to join at every point, so we can sequence the length of the DNA chain we now use fluorescent markers and a computer

• Southern blotting – uses DNA probes to identify complementary DNA sequences after gel electrophoresis. • Northern blotting – Uses DNA to detect RNA species in a similar way to the above. Western blotting is NOT a DNA hybridisation technique - involves the detection of proteins by antibodies after protein gel electrophoresis.

Denature a DNA strand, have a fluorescent probe join to the strand, to identify the sequence that you want can use allele specific probes restriction enzymes to cut DNA DNA gel electrophoresis step to separate fragments of DNA transfer to a membrane hybridisation of a probe to detect a specific piece of DNA and visualisation of the labelled probe

uses mRNA - this tells you what genes are being expressed !

slide with tens of thousands of DNA / genes on it analyse these at the same time take 2 conditions for example Cancer and Normal cells reverse transcriptase mRNA (active, no introns) turns it into cDNA add a flurescent tag Cancer cells labeled red - genes switched on in cancer Normal cells labeled green - genes switched on in normal combine the targets - HYBRIDIZE to microarray cDNA bind at sequence complementary - expression in both types - both bind - red + green = brown only on in cancer - red only on in normal - green we can compare normal person to patient with unknown condition - identify the genetic condition array comparative genome hybridisation

humans DNA differs in non coding DNA use this different mini sattelite loci to identify people in crime scenes and family relatives ect uses a banding pattern identification

look at banding pattern on the chromosome level ! identify male/ female downs syndrome via trisomy on 21 ect

hybridise within a cell, protein will find the complementary sequence chromosome where targeted gene is located will fluoresce - the band will fluoresce we can see small deletions ect