DNA Sequencing Part 1 Flashcards Preview

GN 421 > DNA Sequencing Part 1 > Flashcards

Flashcards in DNA Sequencing Part 1 Deck (46)
Loading flashcards...

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


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


both Maxam-Gilbert and Sanger methods depend on

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


Sanger sequencing

- need primer binding upstream of the region of interest
- 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


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


when are smaller fragments produced

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


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


Depend on DNA synthesis

- Sanger
- Pyro
- Illumina


Depends on Chain termination

- Sanger
- Illumina


Depends on Eelctrophoresis of DNA fragments

- Maxam-Gilbert
- Sanger


Requires making genomic library in a cloning vector

- Maxam-gilbert
- sanger



- next generation sequencing


common features of NGS

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


key of NGS

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


All NGS platforms require

- a library


a library can be obtained by

- amplification
- ligation with custom linkers


each library amplified on a

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


amplification followed by

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


length compared to capillary sequencers

- shorter read length


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


problem with little DNA in a clinical setting

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


hybrid capture

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


probe DNAs

- biotinylated
- selected with streptavidin magnetic beads to purify



- exons of all genes annotated in the reference genome


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


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


what sequencing requires library construction and amplification

- Illumina


3rd generation sequencing

- Pac Bio


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


how pyrosequencing proceeds

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