About Sanger sequencing
- used labelled primers
- uses dideoxynucleotides (H at 2’ and 3’)
- the ddNTPs interrupt DNA synthesis b/c they lack a 3’OH
Sanger Sequencing reaction mixture components
- template DNA (denatured to separate strands)
- single radiolabelled primer
- DNA Polymerase
- dNTPs
- ddNTPs (one base per rxn)
- [dNTPs]»[ddNTPs]
Sanger Sequencing steps
- DNA denaturation (heat)
- Radiolabelled DNA primer annealed to template strand
- four rxn mixtures set up with DNA Pol and free dNTPs
- ddNTPs added to each mixture (only one type in each mixture)
- ddNTPs bind and cease extension
- Each of 4 reactions added to different lane for gel electrophoresis, sequence determined
Dye-terminator Sanger Sequencing Differences
- uses fluorescently-labelled ddNTPs
- 1 rxn mix for all 4 ddNTPs
- uses capillary gel electrophoreisis (still smallest=fastest)
- can read 1000-1500 bp length
Generating template DNA in vitro
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
Generating Template DNA in vivo
-molecular cloning
Molecular cloning steps
- obtain DNA segment to be cloned using restriction endonucleases (they recognize specific sequences)
- Select a carrier molecule that can self replicate (cloning vector) ie. plasmid, bacterial artificial chromosome
- DNA ligase links cloning vector to DNA fragment (now called recombinant vector)
- Host organism provides enzymatic machinery for DNA replication (usually bacteria)
- cloning vector usually has antibiotic resistance so the bacteria with the plasmid can be identified
Components of Plasmid DNA (4)
- Ori (origin of replication
- Restriction sequences
- targets for restriction endonucleases - Small size, allows easy entry into cells
- Antibiotic resistance
- allows selection for calls that contain the intact plasmid or the recombinant plasmid using antibiotics
Next-generation sequencing (NGS)
- 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
Reversible Terminator sequencing Steps
- Library prep
- Cluster Generation
- Sequencing
- Data Analysis
Library prep for RT sequencing steps
- 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)
Cluster Generation for RT sequencing steps
- 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
Sequencing for RT sequencing steps
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
Data analysis for RT sequencing
- 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
