Oligonucleotide Synthesis + Blotting Flashcards Preview

AP0511 Molecular Biology and Genetics > Oligonucleotide Synthesis + Blotting > Flashcards

Flashcards in Oligonucleotide Synthesis + Blotting Deck (17)
Loading flashcards...
1
Q

What is an oligonucleotide?

A

A DNA sequence made up of a ‘few’ nucleotides (3-100 nt)

2
Q

Which direction does DNA get synthesized in phosphoramidite chemistry?

A

3’-5’

3
Q

What is a probe?

A

A small sequence of DNA that is complementary to a specific sequence of target DNA. Can be labelled with a flourescent tag.

4
Q

What are the key parameters that allow for hybridisation of DNA to work?

A
Sequence Concentration
Affinity of probe to sequence
Sequence length
Ionic Strength
pH
Temperature
Mismatches
5
Q

What is the basic calculation for hybridisation melting temperature?

A

2 x (A+T) + 4 x (G+C)

6
Q

What are the 5 types of probes?

A
Genomic DNA
Oligonucleotides
Complementary DNA
Heterologous probe
PCR-generated fragments
7
Q

Explain nick translation labelling?

A

DNase 1 enzyme creates a nick in dsDNA on one strand
DNA exonuclease chews that section of DNA out
DNA pol adds oligo labelled probe at complementary sites (labelled dNTPs)
Heat and anneal, you have a labelled probe

8
Q

Explain random hexamer labelling?

A

dsDNA is heated to form ssDNA
Add hexamer nucleotide probes that will complementary base pair to strand of DNA
Ligase enzyme stitches them together with more ntds
heat and anneal
Probe is formed

9
Q

What are flourochrome probes?

A

Modified nucleotides using enzymes i.e biotinylated dNTP

Used for greater sensitivity

10
Q

How are total proteins detected?

A

Stains like coomassie blue

11
Q

How are specific proteins detected?

A

Using antibodies labelled with enzymes, flourophores, etc

12
Q

What is blotting?

A

Transfer of molecules to a thin membrane for further analysis.
Immobalize and fix on the membrane using heat or UV light.

13
Q

What is low stringency?

A

The conditions of the reaction are not very specific so the probe may not bind specifically to the sequence of DNA.

14
Q

Blotting of DNA is ?
Blotting of RNA is ?
Blotting of proteins is ?

A

Southern blotting
Northern blotting
Western blotting

15
Q

How does DNA move onto the nylon membrane in blotting (3 ways)?

A

Capillary action or by using probes or by using electric (electroblotting)

16
Q

How does blocking reduce background noise?

A

It blocks out the background noise so that the signal can be seen clearer.
By soaking blotting membrane in blocking solution like milk or salmon sperm DNA (something that is not found in your sample)

17
Q

What is the advantage of using microarray gene chip technology over standard blotting?

A

Can analyse 1 sample for 1000s of different probes.

Can detect 4x higher amount of information