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Genomic Sequencing Technologies > Module 2.1 First Generation Sequencing > Flashcards

Module 2.1 First Generation Sequencing Flashcards

1
Q

First Generation Sequencing Strategy

3 steps

A
  • Create fragments of polynucleotide chains
  • Analyze and sort each fragment
  • Assemble
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2
Q

DNA Sequencing using Primer Extension

A
  • 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
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3
Q

General Primer Extension Principle

A

Use synthetic oligos as primers
Binding stability and specificity depend on primer length and sequences
Primer-extension method can be generalized for DNA sequencing

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4
Q

Plus and Minus Technique
(Coulson and Sanger - 1975)

A

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

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5
Q

Maxam-Gilbert Sequencing

A

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

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6
Q

sense strand

A
  • coding strand
  • segment within double-stranded DNA that carries translatable code in the 5’ to 3’ direction
  • has same sequence as mRNA strand
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7
Q

antisense strand

A
  • template strand for the mRNA transcript
  • doesn’t carry the translatable code in a 5’ to 3’ direction
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8
Q

viral RNA genomes

A
  • 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
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9
Q

chromatography

A
  • provides information on the separation and the relative abundance of components
  • can only measure nucleotide composition
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10
Q

mass spectrometry

A
  • provides information on the mass and the structure of individual components
  • can only measure nucleotide composition
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11
Q

RNA as original sequencing target

A
  • easily produced in bulk by culturing microbes
  • single stranded
  • known enzymes that cut RNA at specific sites
  • shorter than DNA molecules
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12
Q

Robert Holly Experiment
(1965)

A
  • 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
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13
Q

Sanger two-dimensional radioactive labeling fractionation procedure

A

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

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14
Q

Sanger two-dimensional radioactive labeling fractionation

two-dimensional paper chromatography

A
  • 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
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15
Q

Sanger two-dimensional radioactive labeling fractionation

auto radiography

A
  • 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
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16
Q

Sanger two-dimensional radioactive labeling fractionation

Benefits

A
  • 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.
17
Q

Sanger two-dimensional radioactive labeling fractionation

scintillation counter

A

instrument for detecting and measuring ionizing radiation

18
Q

DNA sequencing challenges vs RNA

A
  • 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
19
Q

Wu’s primer extension experiment
(conclusions)

A
  • 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
20
Q

Polyacrylamide Gel Electrophoresis (PAGE)

A
  • 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
21
Q

PAGE Gel

urea

A

denaturing reagent that keeps DNA single-stranded while running in PAGE gel

22
Q

phi X 174

A
  • first DNA genome sequenced (1975)
  • single-stranded DNA bacteriophage
  • Plus and minus systems
  • popular positive control genome in labs
23
Q

Plus and Minus system

Difficulties

A
  • 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
24
Q

Maxam-Gilbert Sequencing (1977)

Benefits and Drawbacks

A

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

25
Q

dideoxynucleotide triphosphate (ddNTP)

A

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

26
Q

Sanger chain-terminator sequencing

Original Version

A

-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

27
Q

Sanger chain-terminator sequencing

Benefits

A
  • 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

Genomic Sequencing Technologies (22 decks)

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