Week 2.2: History of Sequencing Technologies Flashcards
(28 cards)
Plus and Minus Technique
(Coulson and Sanger - 1975)
Used PAGE gels
I. Form primer-template duplex
II. DNA synthesis with 32P-labeling (only 1 of 4 bases labeled per synthesis)
-creates double-stranded fragments of various sizes with labeled nucleotides
III. Split into 8 reactions
-4 reactions in the minus system
* dA, dT, dG
* dT, dG, dC
* dG, dC, dA
* dC, dA, dT
-4 reactions in the plus system
* dA
* dT
* dG
* dC
IV. Electrophoresis of 8 reaction products
Bands in + system one base larger than bands in - system
Robert Holley Experiment
(1965)
- able to produce the first whole nucleotide sequence of alanine from yeast
- first isolated pure transfer RNA from yeast
- used the ribonuclease to produce fully and partially digested RNA fragments. Each enzyme cuts the molecule at a specific type of nucleotide.
- determined the composition of the digested fragments using chromatography and mass spec.
- compared pieces from different enzymes and assembled the entire sequence of the yeast tRNA
- developed the clover leaf model tRNA model
Polyacrylamide Gel Electrophoresis (PAGE)
- does a single separation by polynucleotide length only
- Acrylamide polymerization creates a gel matrix with small pores (size controlled by polyacrylamide concentration)
- big molecules will have more resistance from the Gel matrix and travel less distance than small molecules
- usually polymerized between two glass plates
- gel is connected with electrical power with negative and positive electrodes in the buffer systems.
- electrical field is applied across the gel
- nucleic acids are negative charged, so molecules move uniformly away from the negative electrode and towards the positive electrode
- better than 2D chrom for DNA <500 bases long
- allow separation of molecules by one nucleotide
phi X 174
- first DNA genome sequenced (1975)
- single-stranded DNA bacteriophage
- Plus and minus systems
- popular positive control genome in labs
Plus and Minus system
Difficulties
- uneven or lack of generation of sequence fragments during the first round of DNA synthesis
- presence of consecutive rounds of a given mononucleotides
-method is less accurate
dideoxynucleotide triphosphate (ddNTP)
similar to natural deoxynucleotide in the overall structure, except has a 3’ hydrogen instead of 3’ hydroxy group required for extension of DNA chain during synthesis.
-terminates reaction at a specific position where it’s incorporated
Sanger chain-terminator sequencing
Original Version
-DNA denatured to single strands, one used as template
-add DNA primer (radioactively labeled) and DNA polymerase
-split into four reaction tubes with dNTPs and small amount of one ddNTP per tube (all radioactively labeled)
- product of all four reactions separated by electrophoresis in four parallel lanes of PAGE gel
Sanger chain-terminator sequencing
Benefits
- read length and accuracy superior to NGS
-still widely used for smaller scale projects and for validations of deep sequencing results - can produce DNA sequencing reads of >500 nucleotides long with 99% accuracy
Sanger sequencing
Fluorescent dye labeling
- fluorophores were selected to be able to distinguish between the dyes.
- emission wavelengths had to be well resolved from one another
- dyes had to be highly fluorescent to provide sufficient detection sensitivity
- can be either attached to the primer or directly attached to the deoxynucleotides.
fluorescent dye labeled primers
- sample to be sequenced needs to be split into four different reactions, with each reaction using one type of deoxynucleotide and one of the four fluorescent labeled primers
- reaction mixtures are then combined and an electrophoresis down a single polyacrylamide gel. The separated fluorescent bands of DNA can be detected and acquired directly by computer
fluorescent dye labeled ddNTPs
(dye terminators)
- fluorescent dyes are directly attached to the dideoxynucleotide
- Sequencing reaction takes place in a single reaction tube
- Run in a single polyacrylamide gel tube
- separated fluorescent bands of DNA can be detected and acquired directly by computer
capillary gel electrophoresis
procedure
-uses polyacrylamide gel field capillaries
- DNA migrates in capillary from cathode buffer (-) to anode buffer (+)
- separate as they migrate, laser located near output end of capillary excites fluorescent dye of DNA fragment and is read by detector.
- output of detector is sent to computer
- separated DNA fragments appeared as peaks with different migration times
fluorophore
any molecule or functional group which is capable of fluorescence
- all fluorochromes are fluorophores
PCR
Buffer system
solutions that provide suitable chemical environment for optimal activity and stability of the DNA polymerase
Human Genome Project
history
- A large, international scientific effort that generated the first sequence of the human genome and that of selected model organisms
- 20 centers from six countries: US, France, Germany, Japan, China, UK
- Started in 1990, the project was predicted to last fifteen years, with estimated cost of $3B
- initial plan was to finish sequencing the selected model organisms, including yeast, C. elegans, drosophila and mouse and human using automated sequencer
- Celera Genomics, a private company, joined the
race in 1998
shotgun sequencing
history
- first proposed in 1979 for sequencing genomes 4,000- 7,000 bp long
- first genome sequenced was 8000 bp Cauliflower Mosaic Virus (1981) by sequencing 175 individual fragments
Cauliflower Mosaic Virus Shotgun Sequencing
process
- Make large quantity of the virus
- isolate the DNA
- split DNA into multiple reactions
- in each reaction, you treat DNA samples with one restriction enzyme to get a specific set of fragments
- purify the restriction digested DNA fragments and sequence
- identify overlapping regions by looking for the same sequences in two fragments
Whole genome sequencing
main challenge
- if genome has a lot of repeat sequences, it will be hard to identify overlapping regions with high accuracy
- human genome is 3 billion bp long, more than 50% are repeated sequences
Plasmid cloning fragment limit
base pairs
1,000 - 30,000 base pairs
hierarchical genome sequencing
Bacterial Artificial Chromosome
(BAC)
8
- originally created from F’ plasmid.
- able to hold up to 350 KB of DNA
- origin of replication site (ori)
- antibiotic resistance gene
- restriction sites for DNA insertion
- lacZ gene for blue/white colony selection
- present in only one or two copies per cell so able to keep large fragment stable
- Each colony contains particular piece of the genome
coverage
number of times a given nucleotide in a DNA molecule is represented in the library
- quantifies depth or redundancy of representation for a particular genomic region in library
- common QC matrix in genomic sequencing
- need to cover the genome more than one time with redundancy so that you can ensure the proper sequencing of the entire genome region
sequencing coverage
number of times a given position in the DNA is read or sequenced
contig
a set of DNA segments or sequences that overlap in a way that provides a connecting representation of a genomic region
- clone version provides a physical map of a set of cloned segments of DNA across a genomic region
- sequence version provides actual DNA sequence of a genomic region.
- defined by the criteria that each member of a particular subset is related to at least one other member by a significant pairwise overlap within the group
whole shotgun sequencing
method used by Celera Genomics
- Bypass step of building a physical map first, go straight to sequencing genome
- faster and simpler process but more challenging to assemble genome
- Multiple copies of genome are randomly sheared into 2,000 or 10,000 bp pieces and inserted into plasmids for growing in bacteria
- purified plasmids are then subject to Sanger sequencing (pair sequencing)
- two sequences oriented in opposite directions and about the length of a fragment apart from each other were valuable in reconstructing sequence of original target fragments
