Lec 17 - Advanced Molecular Techniques

Question 1

analysing DNA on the neuclotide level - DNA sequencing - how does it work ?

Answer 1

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

Question 2

what are the forms of DNA hybridisation ?

Answer 2

1. Southern blotting – uses DNA
   probes to identify complementary DNA sequences after gel electrophoresis.
2. 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.

Question 3

how does DNA hybridisation work ?

Answer 3

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

Question 4

Explain Reverse Transcriptase PCR

Answer 4

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

Question 5

what is microarray technology ?

Answer 5

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

Question 6

Explain DNA fingerprinting

Answer 6

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

Question 7

what is karyotyping ?

Answer 7

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

Question 8

what is FISH - fluorescent in situ hybridisation

Answer 8

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