Techniques 3: DNA sequencing

Question 1

Describe how you carry out a Mini-prep

Answer 1

Grow lots of bacteria
Break them open
Plasmids small and compact
Genomic DNA is big and tangles and precipitates with other stuff (proteins, lipids etc.)
Results in ug of purified plasmid DNA

Can then use mol bio techniques e.g PCR

Question 2

Describe the process of Gel Electrophoresis

Answer 2

→ Electric current used to separate DNA fragments by size
1. DNA samples loaded into wells + DNA ladder in the gel
2. DNA is -, so when a current is applied to the gel, the fragments migrate to the + end
3. Largest at the top, smallest at the bottom
4. Visualise using a dye which fluoresces under UV light

Question 3

What are some uses of PCR, other than molecular cloning?

Answer 3

• Modern genetic fingerprinting
• Identification of repeat expansions (certain diseases)
• Identification of genomic rearrangements (cancer)

Question 4

What can DNA sequencing be used to check?

Answer 4

Cloning: check no PCR-induced mutations
Sequence in a gene (WT vs mutant)

Question 5

Describe how DNA sequencing is stopped?

Answer 5

Using modified deoxynucleoside triphosphate (dNTP)
Building blocks of DNA synthesis and sequencing

Modified = dideoxynucleoside triphosphate (ddNTP)
No 3’ OH, so once it has been incorporated another cannot bind

Question 6

In DNA sequencing, how do we know how long the DNA mol is?

Answer 6

Gel electrophoresis - discriminate between DNA mols by size

Question 7

In DNA sequencing, how do we know which base is last?

Answer 7

Separate reactions with different ddNTPs:
ddATP, ddTTP, ddGTP, ddCTP

Question 8

Describe the older process of DNA sequencing

Answer 8

• 4 separate reactions, each with a different ddNTP
• Reaction used a radioactively labelled primer
• Agarose electrophoresis of separate reactions - read by hand
• T7 DNA polymerase
• Need a lot of template DNA
• Labour intensive

Question 9

Describe modern automated DNA sequencing

Answer 9

• One reaction contains all ddNTPS - have different fluorescent labels
• Capillary gel electrophoresis - read by computer
• Taq DNA polymerase - PCR like reaction
• More sensitive and more specific
• Quick
• Cheap

Question 10

What is in the master mix for DNA sequencing?

Answer 10

A DNA polymerase
Deoxynucleotides (dNTPs)
Dideoxyonuclotides - flourescently labelled
Buffer

Question 11

What do you need to add to your master mix in DNA sequencing?

Answer 11

Template DNA - plasmid/ PCR product
Primer

Question 12

How can we use restriction enzyme digests to analyse DNA?

Answer 12

different patterns can tell you info about how your molecular cloning went and about different types of DNA