Module 2.1 First Generation Sequencing Flashcards
First Generation Sequencing Strategy
3 steps
- Create fragments of polynucleotide chains
- Analyze and sort each fragment
- Assemble
DNA Sequencing using Primer Extension
- The 12 nucleotide 5’ overhangs at the ends of the linear Lambda DNA
- Used E Coli DNA polymerase to extend and fill the overhangs with different mixes of radio-labeled dNTPs
- Digest DNA fragments with nuclease
- 2D paper chromatography analysis to determine composition
- counted number of nucleotides in single strand region by cutting out 2D chrom spots and measuring with scintillation counter
- omitted various dNTPs to figure out order
General Primer Extension Principle
Use synthetic oligos as primers
Binding stability and specificity depend on primer length and sequences
Primer-extension method can be generalized for DNA sequencing
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
Maxam-Gilbert Sequencing
I. Radio-labelling of DNA fragments
II. Purify single stranded DNA samples
III. Four chemical reactions
* A Guanine/Adenine cleavage (G>A)
* An Adenine-Enhanced cleavage (A>G)
* Cleavage at both Cytosines and Thymines (C+T)
* A cytosine cleavage (C)
IV. Polyacrylamide gel electrophoresis and autoradiograph
Limited by PAGE resolution, can do <400 bases in one round
sense strand
- coding strand
- segment within double-stranded DNA that carries translatable code in the 5’ to 3’ direction
- has same sequence as mRNA strand
antisense strand
- template strand for the mRNA transcript
- doesn’t carry the translatable code in a 5’ to 3’ direction
viral RNA genomes
- virus uses the host cellular machinery to replicate its RNA genome and produce viral proteins
- newly formed viral particles can then go on to infect other cells
chromatography
- provides information on the separation and the relative abundance of components
- can only measure nucleotide composition
mass spectrometry
- provides information on the mass and the structure of individual components
- can only measure nucleotide composition
RNA as original sequencing target
- easily produced in bulk by culturing microbes
- single stranded
- known enzymes that cut RNA at specific sites
- shorter than DNA molecules
Robert Holly 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
Sanger two-dimensional radioactive labeling fractionation procedure
based on detection of radio labeled partial digestion of nucleotide fragments
- grow E. coli in a culture medium containing phosphorus 32
- isolate the 16S ribosome and 23S ribosome
- digest the radio labeled RNA molecules using combinations of ribonuclease.
- fragmented samples separated and analyzed using a two dimensional paper chromatography method.
- paper was dried and position of the separated nucleotide fragments revealed by autoradiograph
- amount of nucleotides could be estimated by intensity of the bands on radio autograph or using counting techniques with a scintillation counter, compared to detecting nucleotides by their absorption of UV light
Sanger two-dimensional radioactive labeling fractionation
two-dimensional paper chromatography
- 1st migration: separates the nucleotides based on their chemical properties such as charge.
- 2nd migration: separates the nucleotides based on a different set of properties such as size or polarity
Sanger two-dimensional radioactive labeling fractionation
auto radiography
- place dried paper containing radio label samples in contact with X ray film, which captures the radioactive signals emitted by the fragmented nucleotides.
- x ray film then provides a visual representation of the separated fragments
Sanger two-dimensional radioactive labeling fractionation
Benefits
- allowed more sensitive detection of nucleotides
- allowed for a more detailed separation of nucleotides, capable of resolving dinucleotides, trinucleotides, and most tetranucleotides in digestive samples prepared by ribonuclease.
Sanger two-dimensional radioactive labeling fractionation
scintillation counter
instrument for detecting and measuring ionizing radiation
DNA sequencing challenges vs RNA
- difficult to obtain a large quantity of homogeneous DNA
- DNA molecules much longer than RNA
- No highly specific DNAses were available at that time for degrading DNA to help sequence analysis
Wu’s primer extension experiment
(conclusions)
- proposed use of specifically designed primers for starting analysis from selected parts of a DNA molecule
- stability of the binding would be determined by length of primers
specificity of the primary template interaction would depend on the sequence uniqueness within the targeting molecules - DNA synthesis could be carried out with controlled incubation time to get labeled fragments for sequence analysis
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
PAGE Gel
urea
denaturing reagent that keeps DNA single-stranded while running in PAGE gel
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
Maxam-Gilbert Sequencing (1977)
Benefits and Drawbacks
Benefits:
- no need to clone DNA
- more accurate than Plus Minus system
- still used for DNA fingerprinting and DNA structure studies
- considered real birth of First Generation Sequencing
Drawbacks:
- large amount of template DNA needed
- use of radioisotopes and hazardous chemicals
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
