Module 3 Section 5 Flashcards

1
Q

About Sanger sequencing

A
  • used labelled primers
  • uses dideoxynucleotides (H at 2’ and 3’)
  • the ddNTPs interrupt DNA synthesis b/c they lack a 3’OH
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2
Q

Sanger Sequencing reaction mixture components

A
  • template DNA (denatured to separate strands)
  • single radiolabelled primer
  • DNA Polymerase
  • dNTPs
  • ddNTPs (one base per rxn)
  • [dNTPs]»[ddNTPs]
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3
Q

Sanger Sequencing steps

A
  1. DNA denaturation (heat)
  2. Radiolabelled DNA primer annealed to template strand
  3. four rxn mixtures set up with DNA Pol and free dNTPs
  4. ddNTPs added to each mixture (only one type in each mixture)
  5. ddNTPs bind and cease extension
  6. Each of 4 reactions added to different lane for gel electrophoresis, sequence determined
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4
Q

Dye-terminator Sanger Sequencing Differences

A
  • uses fluorescently-labelled ddNTPs
  • 1 rxn mix for all 4 ddNTPs
  • uses capillary gel electrophoreisis (still smallest=fastest)
  • can read 1000-1500 bp length
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5
Q

Generating template DNA in vitro

A

If sequence known:
-PCR primers can be designed
If sequence unknown:
-synthetic adapters with known sequences can be ligated to the ends to serve as primer binding regions for PCR

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6
Q

Generating Template DNA in vivo

A

-molecular cloning

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7
Q

Molecular cloning steps

A
  1. obtain DNA segment to be cloned using restriction endonucleases (they recognize specific sequences)
  2. Select a carrier molecule that can self replicate (cloning vector) ie. plasmid, bacterial artificial chromosome
  3. DNA ligase links cloning vector to DNA fragment (now called recombinant vector)
  4. Host organism provides enzymatic machinery for DNA replication (usually bacteria)
  5. cloning vector usually has antibiotic resistance so the bacteria with the plasmid can be identified
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8
Q

Components of Plasmid DNA (4)

A
  1. Ori (origin of replication
  2. Restriction sequences
    - targets for restriction endonucleases
  3. Small size, allows easy entry into cells
  4. Antibiotic resistance
    - allows selection for calls that contain the intact plasmid or the recombinant plasmid using antibiotics
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9
Q

Next-generation sequencing (NGS)

A
  • AKA massively parallel sequencing
  • large DNA segments fragmented into smaller segments (300-400bp), sequenced simultaneously
  • sequences for overlapping fragments aligned to generate consensus sequence for entire DNA segment
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10
Q

Reversible Terminator sequencing Steps

A
  1. Library prep
  2. Cluster Generation
  3. Sequencing
  4. Data Analysis
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11
Q

Library prep for RT sequencing steps

A
  • DNA fragmented into 300-400 bp fragments, adenosine added to 3’ ends to prevent ligation
  • Adapters added (terminal sequences, index sequences, primer binding sequence at 3’ and 5’ ends)
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12
Q

Cluster Generation for RT sequencing steps

A
  • DNA library added to flow cell
  • terminal sequences allow single segments to hybridize with olglionucleotides bound to flow cell
  • DNA Pol extends the olglionucleotide
  • original template DNA washed away
  • adapter sequence at 3’ end of bound DNA hybridizes with nearby olglionucleotide (forms bridge)
  • bridge extended by DNA Pol
  • double strand bridge denatured
  • process repeated, reverse stands hydrolyzed and washed away, leaves unidirectional clonal strands
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13
Q

Sequencing for RT sequencing steps

A

Need following components added:
-fluorescently labelled RT nucleotides added (RT-dATP, RT-dCTP…etc)
-sequencing primer that can hybridize with 3’ adapter region of bound DNA segment
-DNA pol
Process:
-single RT nucleotide added to 3’ end of growing oglionucleotide, unbound nucleotides washed away
-fluorescent signal from cluster is recorded
-fluorescent tag cleaved, nucleotide is unblocked (gives access to 3’-OH)
-repeated for 100-150bp read lengths
-both ends of DNA segment can be read by flipping the bound DNA segment to a nearby oglionucleotide

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14
Q

Data analysis for RT sequencing

A
  • reads are aligned to reference genome
  • if multiple samples pooled together, can be separated by the index sequence
  • read depth is the # of times a specific base pair appears in a sequence read
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