DNA hybridisation and DNA complementarity Flashcards
1
Q
Name some nuclei acid base techniques that are at the heart of complementarity and hybridisation
A
- Northern blotting
- Southern blotting
- Microarrays
- Dideoxy and Next Gen Sequencing
- PCR
- Cloning
2
Q
What is the purpose of hybridisation nucleic acid techniques?
A
- Identifies the presence of NA containing a specific sequence of bases
- Allows the absolute or relative quantitation of these sequences in a mixture
3
Q
What is a probe?
A
-A ssDNA (or RNA) molecule
4
Q
What is the length of a probe?
A
-20 – 1000 bases in length
5
Q
What is the probe labelled with?
A
-Labelled with a fluorescent or luminescent molecule
6
Q
What is the purpose of northern blotting?
A
-Analysis of mRNA or DNA
7
Q
What is the limitation of northern blotting?
A
- Only detects one gene at a time and small numbers of samples
- The gel based techniques are Time consuming and messy
8
Q
What is northern blotting superseded by?
A
-Quantitative PCR or other techniques
9
Q
Describe the northern blotting process
A
- Uses DNA or RNA respectively that is separated by gel electrophoresis
- Then transferred by mass capillary flow of a buffer from a reservoir to a nylon membrane carrying the nucleic acid with it
- It is captured by and covalently bonded to the membrane and then hybridised with a labelled probe
10
Q
What is the modern day process of northern blotting?
A
- Electroblotting where the gel is sandwiched between electrodes and a voltage is applied to two electrodes and the negatively charged DNA or RNA is transferred to the membrane electrophoretically
- Again the DNA or RNA is captured by and covalently bonded to the membrane and then hybridised with a labelled probe
- The probe can be visualised by some means
11
Q
What is the purpose of microarrays?
A
- To analyse the expression of thousands of transcripts in each sample.
- See which genes are expressed
- When why and under what conditions
12
Q
Describe the process used with microarrays
A
- For this RNA is extracted
- Labelled usually with a fluorescent molecule
- Hybridised to the array and the amount and location of the label measured
- We can then compare these measurements and determine changes in specific genes, identify signatures that relate to specific diseases or conditions
- This tells us how much of each and everyone of the transcripts in the human genome are being expressed and is an alternative to RNASeq, a next generation sequencing alternative that you will also learn about
