Tool Kit

Question 1

What are restriction nucleases

Answer 1

Function-
Recognise specific DNA sequence and cleaves the DNA (defense mechanism against foreign DNA)

They are found in bacteria and archae (EcoR1, Hind111, BamH1)

Question 2

What are ligases

Answer 2

Function-
Catalyse the joining of DNA strands through phosphodiester bonds

They are used in DNA cloning and PCR (T4)

Question 3

What are kinases

Answer 3

Function-
Catalyse the transfer of phosphate groups to molecules (uses ATP)

they are used in DNA probing (T4 DNA ligase)

Question 4

What are phosphatases

Answer 4

Function-
Removes phosphate groups from molecules

Used to prevent self ligation

Question 5

What are DNA polymerases

Answer 5

Synthesises DNA molecules. Thermostable ones like taq used in PCR

Question 6

What are Reverse transcriptase

Answer 6

Catalyses DNA from RNA template
Found in retroviruses and used in cDNA synthesises

Question 7

What are RNA polymerases

Answer 7

Catalyses RNA from DNA template

Question 8

What are exonucleases

Answer 8

Cleaves nucleotides at the end of the polynucleotide chain

Question 9

What are RNases

Answer 9

Catalyses the degradation of RNA into smaller components
Used in RNA cleanup or in vitro transcription

Question 10

How does gel electrophoresis work

Answer 10

Prepare the gel with agarose (separation of wide range of fragment sizes) or acrylamide (operates 1-1000 BP) and a comb is used to create wells

Buffer is added

Load samples onto wells with a stain (sybr green) along with DNA ladder

Electrical current applied and samples visualised under UV

Question 11

What are the blotting techniques

Answer 11

Southern-
Targets DNA by transferring gel onto a membrane and hybridising it with labeled DNA probe complementary to target sequence

Northern-
Targets RNA by transferring gel to membrane with labelled RNA probe (can help assess RNA levels and gene expression)

Western-
Targets proteins by transferring gel to membrane with specific antibodies that bind to target protein which are then detected using a secondary antibody labelled with an enzyme or flurophore

Question 12

Difference between liquid and solid phase hybridisation

Answer 12

Solid hybridisation is immobilising nucleic acid strand on solid support (membrane) so you can remove unreacted probes but you add a diffusion barrier which decreases reaction rate

Liquid phase is hybridisation in liquid solutions (microRNA detection) and is used when reaction rate is priority