DNA sequencing

Question 1

DNA polymerase needs a primer so what could you do if you don’t know the DNA sequence?

Answer 1

Random hexamers- not very specific
When cloning in a vector, use known sequence of target DNA with restriction enzymes & ligase- so primer binds to vector DNA but moves along inserted DNA

Question 2

What is dideoxynucleotide (Sanger) DNA sequencing used for?

What are the steps?

How could this be improved?

What’s the difference between dNTP and ddNTP?

Answer 2

Determine nucleotide sequence of DNA

1. 4 reactions containing same single strand target DNA, 4 deoxyribonucleoside triphosphates, DNA polymerase, primer & 1 type of dideoxyribonucleoside triphosphate (ddNTP) - all mixed
2. Add nucleotides to 3’ end of primer with target DNA as template
3. When ddNTP (radioactively labelled) incorporated synthesis terminates (bc lacks 3’ OH) so synthesis terminates lots different strands = make lots DNA fragments different lengths with different ddNTP of same base
4. Fragments are separated by gel electrophoresis
5. Autoradiograph & read bands- sequence obtained is complementary to original template (5’ at bottom of gel)

Tag the ddNTPs in different fluorescent dye- so fragments end in same colour if have same ddNTP attached- when load onto gel electrophoresis detect colour with laser beam & detect what base present on computer peak readout & find the complimentary sequence.

dNTP has 3’ OH, ddNTP has 3’ H

Question 3

Next generation pyro sequencing

What are the 8 steps involved?

How are the phosphates catalysed? How does this produce light?

How many nucleotides can pyrosequencing read in read length?

Answer 3

1. Hybridise target DNA into double strand fragments
2. attach adapters with primer sequences to each fragment to make DNA single stranded.
3. Attach each DNA fragment to bead surround with solution for reagents for PCR
4. amplify fragments w/PCR
5. each bead forced into well & layered with sequencing reagents so DNA synthesis occurs in each well
6. Add solution with specific dNTP over plate
7. When nucleotide added to growing chain-releases PPi to produce light emitting chemical reaction (some nucleotides when added won’t)
8. Amount of light proportional to number of nucleotides added

By ATP Sulphurylase- into ATP to use by luciferase to catalyse luciferin & release flash light

500

Question 4

What is shotgun sequencing?

What is a contig?

Sequencing depth?

Deep sequencing?

Answer 4

DNA fragments are assembled in computer programs to find where they overlap to determine order of DNA bases

Length that can be read by overlapping fragments (~1 mil bp)

No. times a particular nucleotide is sequencing on average (~10)

Amount DNA present increased to allow sequencing depth of x100/x1000 - informative for under represented mutations/variation