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GN 421 > DNA Sequencing Part 1 > Flashcards

DNA Sequencing Part 1 Flashcards

1
Q

how to make a genomic library

A
  • DNA isolated from cells
  • restriction enzymes to cleave DNA or cleave from vector
  • insert into recombinant plasmid
  • transform bacteria
  • grow transformed bacteria to make a genomic library containing all DNA fragments in the genome
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2
Q

Maxim-Gilbert Chemical sequencing

A
  • does not involve DNA synthesis
  • uses chemical treatment that breaks DNA chain after G, A+G, C+T, C
  • different chemicals to get cleavage after different sites
  • label fragments at 5’ end. separate out on gel
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3
Q

both Maxam-Gilbert and Sanger methods depend on

A
  • separation of labeled DNA fragments by electrophoresis

- limits sequencing long stretches of DNA

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4
Q

Sanger sequencing

A
  • need primer binding upstream of the region of interest
    (template)
  • DNA polymerase will add on the complementary nucleotide
  • to determine the exact sequence, the reaction can be stopped using terminators
  • dideoxy sequencing or chain termination
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5
Q

Sanger Dideoxy Chain termination

A
  • ssDNA, DNA pol, all ddNTP’s, labeled primer, template DNA
  • 4 tubes containing all the components needed to polymerize DNA - adds a small amount of ddNTP to each tube
  • each tube also contains one ddNTP at 1/100 the concentration of dNTPs
  • these have no hydroxy at the 3’ end and thus another NTP cannot be added to them - chain terminators
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6
Q

when are smaller fragments produced

A
  • when ddNTP added closer to primer

- chains are smaller and migrate faster

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7
Q

how to measure Sanger

A
  • creates a pool of DNA sequences of different length all ending with that specific nucleotide
  • terminates reaction at every A, T, G, and C nucleotide in each tube.
  • run on 4 lanes and visualize
  • shortest bands travel furthest
  • will emit light at different wavelengths
  • camera detects DNA
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8
Q

Depend on DNA synthesis

A
  • Sanger
  • Pyro
  • Illumina
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9
Q

Depends on Chain termination

A
  • Sanger

- Illumina

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10
Q

Depends on Eelctrophoresis of DNA fragments

A
  • Maxam-Gilbert

- Sanger

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11
Q

Requires making genomic library in a cloning vector

A
  • Maxam-gilbert

- sanger

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12
Q

NGS

A
  • next generation sequencing
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13
Q

common features of NGS

A
  • sample preparation
  • sequencing machines - solid surface
  • data output
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14
Q

key of NGS

A
  • massively parallel sequencing reactions

- capable of analyzing millions, even billions of reactions at a time

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15
Q

All NGS platforms require

A
  • a library
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16
Q

a library can be obtained by

A
  • amplification

- ligation with custom linkers

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17
Q

each library amplified on a

A
  • solid surface with covalently attached adapters that hybridize the library adapter
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18
Q

amplification followed by

A
  • direct step-by-step detection of nucleotide base incorporated by each amplified library fragment set
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19
Q

length compared to capillary sequencers

A
  • shorter read length
20
Q

library construction and amplification

A
  • shear high molecule weight DNA with signification
  • polish ends to make blunt
  • ligate synthetic DNA adapters via PCR
  • produce size fractions via PCR
  • quantitate
  • amplify library fragments on flow cell surface using PCR
  • denature clusters to single-stranded
  • hybridize sequence primers to linearized ss cluster DNAs
  • proceed to sequencing or hybrid capture
21
Q

problem with little DNA in a clinical setting

A
  • polymerase errors problems early

- PCR amplify high/low GC content less well than 50% GC content

22
Q

hybrid capture

A
  • fragments from whole genome library are selecting by combining with probes that correspond to most human exons or gene targets
23
Q

probe DNAs

A
  • biotinylated

- selected with streptavidin magnetic beads to purify

24
Q

exome

A
  • exons of all genes annotated in the reference genome
25
Q

how hybrid capture works

A
  • target part of genome of interest
  • probes hybridize to exons
  • magnetic field to capture biotinylated DNA beads pull down hybridized fragments and get rid of rest of library
  • denature away DNA you want
  • library you’re sequencing is much reduced in complexity
26
Q

multiplex PCR amplification of Targets

A
  • if you want a very small subset of a genome
  • amplify genes of interest first
  • then make sequencing library
  • then sequence
27
Q

what sequencing requires library construction and amplification

A
  • Illumina
28
Q

3rd generation sequencing

A
  • Pac Bio
29
Q

Pyrosequencing 454

A
  • reaction monitored by the release of a pyrophosphate during each nucleotide incorporation
  • the released pyrophosphate is used in a series of chemical reactions in the generation of light
  • light emission detected by a camera which records the appropriate sequences
30
Q

how pyrosequencing proceeds

A
  • incubating one base at a time
  • measuring the light emission
  • degrading unincorporated bases
  • addition of next base
31
Q

advantages of pyrosequencing

A
  • large read lengths

- comparable to sanger sequencing

32
Q

disadvantages of pyrosequencing

A
  • high reagent costs

- high error rate over strings of 6+ homopolymers

33
Q

sequencing by synthesis

A
  • utilizes the step incorporation of reversibly fluorescent and terminated nucleotides for DNA sequencing
  • all 4 labeled nucleotides are added to the sequencing chip at the same time and one sticks
  • remaining washed away
  • fluoro signal read
  • then cleaved and washed away
  • repeated until process complete
34
Q

example of sequencing by synthesis

A
  • Illumina
35
Q

advantage to sequencing by synthesis

A
  • overcomes homopolymers issue due to terminated nucleotides
36
Q

disadvantage to sequencing by synthesis

A
  • increased error rate with increased read lengths
  • failure to completely remove fluorescence
  • increasing background noise
  • chemistry is never 100%
37
Q

Ion semiconductor sequencing

A
  • utilizes the release of H+ ions from the sequencing reaction to detect the sequence of a cluster
  • each cluster located directly above a semiconductor transistor which is capable of detecting changes in pH in the solution
  • During nucleotide incorporation, a single H+ ion is released into the solution and detected by a semiconductor
38
Q

advantages of ion semiconductor sequencing

A
  • more cost effective and time efficient
  • low substitution error rate
  • improved analysis
39
Q

disadvantages of ion semiconductor sequencing

A
  • not paired-end
  • insertion/deletion
  • homopolymer problems
40
Q

Pac Bio sample prep

A
  • shearing
  • polish ends
  • SMRTbell ligation
  • sequencing primer annealing
41
Q

Pac Bio Library/Polymerase complex

A
  • DNA pol binding

- load library/pol energetics onto SMRT

42
Q

Pac Bio Sequencing

A
  • raw reads
  • post filter reads
  • mapped reads
  • each of four nucleotides is labeled with a different colored fluorophore
  • diffuse in and out
  • if they dwell long enough will get detected.
43
Q

Advantages to Pac Bio

A
  • true single molecule sequencing rather than clusters
  • polymerase adhered to bottom of well to pinpoint active site with objects of machine to detect sequencing reaction
  • allow for longer read lengths
44
Q

Nanopore

A
  • when a small voltage is imposed across a nano pore in a membrane separating two chambers containing aqueous electrolytes, the ionic current through the pore can be measured
  • molecules gong through the nano pore cause disruption of the ionic current
  • by measuring the disruptions molecules can be identified.
45
Q

advantage of nanopore

A
  • use small amount of DNA
  • sequence on site rapidly
  • true reagentless sequencing
46
Q

disadvantage of nanopore

A

-challenge to get uniform pores

GN 421 (36 decks)

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