DNA sequencing and Barcoding

Question 1

DNA polymerase

Answer 1

• copies DNA in 5’ - 3’ direction from the primer

Question 2

helicase

Answer 2

unwinds DNA

Question 3

ddNTPs, Sanger sequencing

Answer 3

• dideoxyribonucleotides, modified base
• extra oxygen
• DNA polymerase incorporates them into strand
• terminates strand
• Sanger sequencing incorporates small number of fluorescently tagged ddNTPs, so a termination product for every base position is determined
• electrophoresis can then sort the strands into size order and the fluorescently tagged ddNTPs are read, showing the sequence of DNA

Question 4

electropheresis

Answer 4

• DNA is negatively charged so moves through a gel matrix towards a positively charged electrode when a current is applied
• smallest molecules move the fastest

Question 5

capillary sequencing

Answer 5

• termination products are run through a capillary gel to sort into size order past a laser/detector
• as each product passes by the colour peak at each of the 4 wavelengths is recorded
• the computer measures the intensity of the colours and decides
  which base was present given the ratio of the 4 colours (basecalling, not always accurate)
• creates a sequence “trace” or chromatogram

Question 6

DNA barcoding

Answer 6

• all organisms within a branch of evolutionary tree share common genes
• can design universal primers that fit these common genes where the sequence within primers differs enough to determine species
• relies on having accurate databases of sequences from each
  species

Question 7

specific DNA barcodes

Answer 7

• RbcL, plants
• CO1, animals
• ITS1, plants/animals/fungi
• sometimes multiple are needed to distinguish between organisms

Question 8

next generation sequencing, massively parallel sequencing

Answer 8

• millions of short DNA fragments can be sequenced simultaneously, quicker than Sanger sequencing
  1. Fragment the DNA into short stretches
  2. Bind on adaptor sequences – These have a recognisable code a
  priming region and a sequence complementary to those a flow
  cell.
  3. DNA fragments bind to the physical surface of a flow cell - a
  glass slide or microscopic bead.
  4. The computer records the position of each individual DNA
  fragment
  5. Sequencing reactions are performed, recording
  the sequence for each one of millions of molecules at the same
  time
• e.g. Illumina sequencing

Question 9

illumina sequencing

Answer 9

• similar to Sanger sequencing, involves chain termination
• reversible, once recorded the fluorescent tag is cleaved off and washed away
• the next base can then be added
• requires less DNA, can just use template molecule

Question 10

flow cells

Answer 10

• used in NGs
• hollow glass slides
• single stranded DNA in liquid solution pumped in
• DNA binds to surface of flow cell and is replicated to form cluster of clonally replicated DNA templates
• computer records colour change and sequences them in parallel

Question 11

metabarcoding

Answer 11

• barcoding mixed DNA from several species that cannot be isolated
• separates DNA out on a surface so different samples do not overlap
• using Miseq NGS, generates slightly longer base pairs sequences so more suited to barcoding
• used in medical studies of bacterial populations in patients, pollinator studies in plants, pollen tracking, detecting contamination/fraud in meat trade

Question 12

single molecule sequencing

Answer 12

• lots of individual cells with a nanopore at
  the bottom of each wide enough to let a single base through
• enzyme bound to the pore processes each base in turn, generating signal that can be read to generate sequence
• doesn’t rely on amplification or adaptor ligation, should be able
  to get much longer read lengths