DNA Sequencing Part 1 Flashcards Preview

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Flashcards in DNA Sequencing Part 1 Deck (46):
1

how to make a genomic library

- 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

2

Maxim-Gilbert Chemical sequencing

- 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

3

both Maxam-Gilbert and Sanger methods depend on

- separation of labeled DNA fragments by electrophoresis
- limits sequencing long stretches of DNA

4

Sanger sequencing

- 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

5

Sanger Dideoxy Chain termination

- 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

6

when are smaller fragments produced

- when ddNTP added closer to primer
- chains are smaller and migrate faster

7

how to measure Sanger

- 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

8

Depend on DNA synthesis

- Sanger
- Pyro
- Illumina

9

Depends on Chain termination

- Sanger
- Illumina

10

Depends on Eelctrophoresis of DNA fragments

- Maxam-Gilbert
- Sanger

11

Requires making genomic library in a cloning vector

- Maxam-gilbert
- sanger

12

NGS

- next generation sequencing

13

common features of NGS

- sample preparation
- sequencing machines - solid surface
- data output

14

key of NGS

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

15

All NGS platforms require

- a library

16

a library can be obtained by

- amplification
- ligation with custom linkers

17

each library amplified on a

- solid surface with covalently attached adapters that hybridize the library adapter

18

amplification followed by

- direct step-by-step detection of nucleotide base incorporated by each amplified library fragment set

19

length compared to capillary sequencers

- shorter read length

20

library construction and amplification

- 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

problem with little DNA in a clinical setting

- polymerase errors problems early
- PCR amplify high/low GC content less well than 50% GC content

22

hybrid capture

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

23

probe DNAs

- biotinylated
- selected with streptavidin magnetic beads to purify

24

exome

- exons of all genes annotated in the reference genome

25

how hybrid capture works

- 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

multiplex PCR amplification of Targets

- if you want a very small subset of a genome
- amplify genes of interest first
- then make sequencing library
- then sequence

27

what sequencing requires library construction and amplification

- Illumina

28

3rd generation sequencing

- Pac Bio

29

Pyrosequencing 454

- 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

how pyrosequencing proceeds

- incubating one base at a time
- measuring the light emission
- degrading unincorporated bases
- addition of next base

31

advantages of pyrosequencing

- large read lengths
- comparable to sanger sequencing

32

disadvantages of pyrosequencing

- high reagent costs
- high error rate over strings of 6+ homopolymers

33

sequencing by synthesis

- 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

example of sequencing by synthesis

- Illumina

35

advantage to sequencing by synthesis

- overcomes homopolymers issue due to terminated nucleotides

36

disadvantage to sequencing by synthesis

- increased error rate with increased read lengths
- failure to completely remove fluorescence
- increasing background noise
- chemistry is never 100%

37

Ion semiconductor sequencing

- 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

advantages of ion semiconductor sequencing

- more cost effective and time efficient
- low substitution error rate
- improved analysis

39

disadvantages of ion semiconductor sequencing

- not paired-end
- insertion/deletion
- homopolymer problems

40

Pac Bio sample prep

- shearing
- polish ends
- SMRTbell ligation
- sequencing primer annealing

41

Pac Bio Library/Polymerase complex

- DNA pol binding
- load library/pol energetics onto SMRT

42

Pac Bio Sequencing

- 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

Advantages to Pac Bio

- 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

Nanopore

- 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

advantage of nanopore

- use small amount of DNA
- sequence on site rapidly
- true reagentless sequencing

46

disadvantage of nanopore

-challenge to get uniform pores