Module 5.2 Emerging NGS Platforms Flashcards
Phi29 DNA Polymerase
- enzyme from bacteria phage Phi29
- simple monomer protein
- C-terminal domain: polymerase
- N-terminal domain: strong 3’ to 5’ exonuclease activity
Exceptionally High processivity: - capable of incorporating more than 10,000 nucleotides in a single binding event, one of the most processive known
Strong proof reading activity: - may produce 1-2 orders of magnitude less error than Taq polymerase
- Highly processive
- can degrade single stranded oligonucleotides without dissociation
Remarkable Strand-displacement activity - acts similar to helicase through strong binding to single stranded DNA.
- optimal temp = 30 degrees (isothermal)
Strand displacement Amplification
ability to displace or push aside a downstream complementary DNA strand while synthesizing DNA using another strand as template in the 5’ to 3’ direction.
Multiple Displacement Amplification
(MDA)
process
- multiple random hexamer primers bind to DNA template
- strand displacing polymerase (Phi29) amplifies from primer binding sites.
- extension progresses in 5’ to 3’ direction
- Phi29 displaces newly synthesized strand on 3’ side and creates single stranded branches that can bind new random hexamer primers
- primers and polymerase continue to bind and amplify on branched strands
- end results >30 KB and highly branched networks of DNA
Rolling Circle Amplification
(RCA)
single primer process
- primer hybridizes to template at target DNA sequence
- strand-displacement DNA polymerase (eg. Phi29) extends primer along circle template and displaces newly synthesized strand to continues making new strand
- process continues in a rolling circle manner forming of a long single stranded DNA product composed of repeated units of original circular template.
- resembles a tandem array of the target sequence
tandem array
A series of copies of a gene arranged one after another along a chromosome
Rolling Circle Amplification
(RCA)
multiple primer process
- can use multiple primers (random or target-specific) that are complementary to forward and reverse strands
- primers amplify circular DNA in one direction and displace newly synthesized strand
- displaced single strands amplified by primers in other direction like MDA
- bidirectional amplification continues to produce long and highly branched double strand DNA with repeat units
Complete Genomics
Library preparation
adapter features
- Read 1 and 2 adapters bind together to form D-loop structure
- Read 1= 5’ stem -> D loop -> T overhang 3’
- Read 2= 3’ overhang-> sample index -> stem -> phosphate group 5’
Complete Genomics
Library Preparation
process
- target DNA fragmented to proper size and end-repaired to achieve the blunt end with 5’ phosphate group and A-tailing at 3’ end
- ligate adapters to ends of target molecule to create library molecules
- PCR forward primer matches loop, reverse primer matches 3’ overhang
- create the double stranded read 1 adapter, dna fragment, read 2 adapter with sample index
DNA Nano Balls
(DNB)
process
- library molecules denatured to create two single stranded molecules.
- forward strand used to form circular DNA
- single stranded library molecule annealed to splint molecule that brings two ends of library molecules together, forming short stretch of double strands with a nick.
- nick is sealed by ligase to form a closed circle.
- splint molecules and non ligated linear molecules removed using DNA exonuclease
- single strand DNA circle replicated with Phi29 polymerase via RCA using primer that specifically binds to adapter sequence
- Generates long single stranded DNA with hundreds of tandem copies of library molecule
- Under proper buffer conditions, DNA folds into 3D ball about 220 nm in diameter in solution
splint molecule
a single stranded, short sequence that is complementary to 5’ and 3’ end of adapter
DNB
DNA exonuclease
Enzyme which specifically digests linear DNA without cutting circular DNA
DNB
Rolling Circle Amplification
(RCA)
benefits and drawbacks
Benefits
- linear molecules removed by exonuclease minimize the index hopping risk
- no polymerase dissociation and reassociation and Phi29 high processivity minimizes risk of truncation during clonal amplification.
- higher fidelity amplification through continuous copying of the original circular DNA template. Avoids creation of clonal errors like in PCR
Drawbacks
- small insert fragment size (<700bp, 500bp preferred)
- longer inserts are harder to ligate to form a circle and fewer copies within DNA ball.
- Too few copies in DNA ball = weak signal. Copy number limited by insert size and reaction time
DNB
Patterned Array
features
- solid surface with grade patterned arrays of positively charged spots fabricated via photolithography, etching techniques then chemical modification to generate a sequencing flow cell.
- One spot = 300 nm diameter,separated by 700 nm from center to center to allow easy attachment of DNB to flow cell.
- increases DNA content per array and image information density relative to non pattern arrays
Cool MPS
features
- no fluorescently labeled reversibly terminated nucleotides required
- uses unlabeled nucleotides with reversible termination group at the 3’ carbon position, which will pause the DNA polymerase upon incorporation into growing chain
- base determination via fluorescently labeled antibodies
Cool MPS
process
- Sequencing primers bind to hundreds of adapters
- DNA polymerase adds 1 of 4 unlabeled, reversibly terminating nucleotides to 3’ end of primer
- unbound nucleotides are washed away, and DNA synthesis is paused by blocking group on 3’ end.
- 4 types of fluorescent labeled base-specific monoclonal antibodies that only bind bases with blocking groups are added to flow cell
- unbound antibodies are washed away
- fluorescent signal captured by camera and converted to digital output for base calling
- bound antibodies and blocking groups removed to regenerate natural nucleotides with no scar to allow longer extension
- cycle repeated
