Cloning and sequencing Flashcards
What is Gibson Assembly (GA) ?
A novel method for the easy assembly of multiple linear DNA fragments. Regardless of fragment length or end compatibility, multiple overlapping DNA fragments can be joined in a single isothermal reaction. With the activities of three different enzymes, the product of a Gibson Assembly is a fully ligated double-stranded DNA molecule.
What are the steps involved ?
- Forming 3’ single stranded overhangs –> T5 exonuclease, but unstable at 50 ̊C and exposes 3’ ends after some time
- Annealing complementary termini
- Gap-filling –> Phusion High Fidelity DNA polymerase
- Nick-sealing –>Taq DNA ligase
What are the advantages of GA ?
- Gibson assembly for cloning multiple fragment
- Simultaneous assembly of up to 8 -10 fragments
- Fragments can be short (=annealed oligos without PCR amplification)
- Vector can be either PCR-amplified or linearized by restriction enzymes
Which biological macromolecule was sequenced first?
Protein (Primary structure of insulin 1949-1951)
Proteins are made of linear polypeptides
“They (the proteins) seem to be put together in an order that is random, but nevertheless unique and most significant, since on it must depend the important physiological action of the hormone.” (from the acceptance speech for the Nobel prize 1958)
What was the implication of the double helical structure (1953) for base sequences in DNA ?
- Structure places no constrain on sequence
- Suggests mechanism for faithful replication
- Stage set to solve the “coding problem” (“Coding problem” solved before any DNA sequence was experimentally determined)
What were the major challenges during the early days of sequencing ?
- Different DNA molecules were chemical very similar separation difficult
- Chain length of DNA much greater than for protein, complete sequencing seemed unapproachable
- Amino acids have widely varying properties. only 4 bases for DNA
- No base-specific DNAses were known
- Protein sequencing has depended upon specific proteases
What was the first DNA sequence obtained ?
By which method ?
Lambda cos ends – 12 bases – partial 1968 – complete 1971
Method: repair reaction from 3’OH end (E. coli polymerase) using radioactive nucleotides followed by partial nuclease degradation - isolation of the synthesized oligonucleotide - sequence determination
What are the advantages/disadvantages of “Plus and Minus” Sanger sequencing (1975) ?
Advantage: - Rapid and allowed sequencing phiX174 Disadvantage: - Single stranded DNA - Accuracy --> 8 reactions (4 for plus and 4 for minus in parallel) --> confirmatory data
What are the advantages/disadvantages of the “Maxam and Gilbert” sequencing method (1977) ?
Advantage: - Double stranded DNA - 4 reactions are sufficient Disadvantage: - Strand separation
What are the advantages/disadvantages of chain termination or dideoxy sequencing (Sanger sequencing – 1977) ?
Based on finding from Atkinson et al. (1969) that ddTTP can inhibit DNA polymerase I
Advantage:
- Increased accuracy
- 4 reactions (only)
Disadvantage:
- ssDNA (phage M13 later also alkaline denaturation)
What are the requirements for chain-termination sequencing of DNA ?
Single-stranded DNA molecule (template) to be sequenced
Oligonucleotide (primer) complementary to upstream region of template DNA polymerase
DNA synthesis reaction is performed
- Primer/template mix distributed to four tubes
- 4 dNTPs plus DNA polymerase
- a radioactive dNTP in one of four tubes add ddATP
- in a second add ddCTP
- in the third ddGTP
- in the fourth ddTTP
By adjusting the ratio of dNTP to ddNTP it is possible to generate the full spectrum of terminated products with approximately equal representation. How ?
- too much ddNTP gives preferentially short products
- too little ddNTP gives preferentially long products
Fo Sanger sequencing, how are the reaction products denatured and electrophoresed ?
How are bands detected ?
How many nt could be read per 4 lanes ?
- reaction products are denatured by adding formamide and heating to separates the newly synthesized radioactive strands from the template
- samples are loaded on to a thin polyacrylamide gel
containing urea and separated by electrophoresis at high voltage (~2500 V) - the thin gels allow a high resolution of DNA molecules:
one base different in length - plates can be heated to keep the DNA denatured
- gel is fixed and dried and exposed to X-ray film to reveal the chain terminated products as bands
Sequencing reads are initially ~100 nt per 4 lanes which increased with improvements to the label (35S replaced 32P) and the gel (wedged, longer gels, shark tooth comb) system to ~350 nt
Why was dye terminator sequencing more advantageous than conventional Sanger sequencing ?
Automation of DNA sequencing (Hood Caltech & Applied Biosystems (ABI) Initially a primer was fluorescently end labelled with 4 different dyes –> different primer was used in each of the 4 dideoxy sequencing reactions
This was later replaced by the dye-terminator sequencing, where each of the dideoxy nucleotides is labelled with a different coloured fluorescent tag - one reaction
Sequencing reactions are performed in a PCR machine in a technique called cycle sequencing
What were the advantages of automating the dye terminator sequencing method ?
- Visualization of sequences obtained with dye-termination
- Increase the number of lanes up to 96
Increase length of gel - Bioinformatic: “gel tracking” & sequence extraction
- Replacing the gel by capillary electrophoresis
- Separation by size based on their total charge (5-20 kV)
- Polymer solution replaces need of manually poured polyacrylamide gels
- Automatic sample loading (Electrokinetic injection)
- ABI Prism 3700 with 96 capillaries (1998) produced read length of up to 1000 nt per capillary per run
