SCIE1106 Lecture Notes - Lecture 17: In Situ Hybridization, Northern Blot, Southern Blot
L17 Hybridisation Techniques
•
Describe the conditions under which ds nucleic acid
molecules are denatured/renatured
•
Know that hybridisations occur between RNA/RNA or
DNA/RNA
•
Describe differences between heterologous/
homologous probes
•
Describe Southern, Northern and in situ hybridsiation
techniques and give examples of the uses of these
techniques
DNA is all about DNA denaturation & renaturation
Hybridization may occur between
1. Complementary RNA strands (called double
stranded RNA molecules)
2. Complementary DNA & RNA strands (called
RNA/DNA hybrids)
Probes?
• 15-1000 nt long
• a piece of DNA with known sequence
• works with formamide, ssDNA binds together
to form dsDNA if theres high complementary
Homologous probes – are used to detect identical
nucleic acid molecules
Heterologous – are used to detect related molecules
How? What Techniques?
1. Southern blotting
a. Hybridization between DNA probe +
DNA target
2. Northern blotting
a. Hybridization between DNA probe +
RNA target
3.
In situ
hybridization
To begin with 1 of the 3 techniques, DNA molecules
need to be labelled → Random priming
Label nucleotides of probes, with
1. Chemicals – Digoxygenin probe
a. Spacer exists to let dig stick out when
double helix forms
2. Radioactive - 32P at alpha P of ATP
Requirements to label DNA probe
1. DNA polymerase to incorporate labelled nt
2. Hexanucleotides ( 6nt, random sequence) as
primers to initiate sequencing
3. Labelled dNTP
4. DNA Probe
When the sequence is labelled we can track where it
binds/ what it binds to
find more resources at oneclass.com
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Document Summary
L17 hybridisation techniques: describe the conditions under which ds nucleic acid molecules are denatured/renatured, know that hybridisations occur between rna/rna or. Dna/rna: describe differences between heterologous/ homologous probes, describe southern, northern and in situ hybridsiation techniques and give examples of the uses of these techniques. Dna is all about dna denaturation & renaturation. Hybridization may occur between: complementary rna strands (called double stranded rna molecules, complementary dna & rna strands (called. Probes: 15-1000 nt long, a piece of dna with known sequence, works with formamide, ssdna binds together to form dsdna if theres high complementary. Homologous probes are used to detect identical. Heterologous are used to detect related molecules nucleic acid molecules. What techniques: southern blotting, hybridization between dna probe + Dna target: northern blotting, hybridization between dna probe + To begin with 1 of the 3 techniques, dna molecules need to be labelled random priming.
