LO3: Development of DNA Sequencing Technologies

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46 Terms

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Molecular Biology

a field of biology that studies the composition, structure, and interactions of cellular molecules that carry out the biological processes essential for the cell’s function and maintenance

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Central Dogma

Illustrates the flow of genetic information from DNA to RNA to Protein

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DNA Replication

process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules

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Steps of DNA Replication

Unwind, Prime, Elongate

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DNA Polymerase

The enzyme responsible for constructing new DNA strands during replication or DNA repair.

Can only elongate DNA where there is a free 3’ -OH group that it can act on.

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DNA Helicase

The enzyme that unwinds (unzip) the DNA molecule near the replication fork

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Single-Stranded DNA-Binding Proteins (SSBs)

Are proteins that bind to and stabilize the single-stranded regions of DNA that result from the action of unwinding protein

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Topoisomerase

It facilitates DNA replication by reducing molecular tension caused by supercoiling upstream from the replication fork (e.g. DNA gyrase)

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Primase

Is a polymerase that initiates replication by synthesizing short segment of RNA as source of the 3’-OH end that DNA polymerase can use.

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DNA Ligase

It seals nicks or breaks in the sugar-phosphate backbone generated during replication

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Polymerase Chain Reaction

a laboratory technique generating tens of billions of copies of a particular DNA fragment (the sequence of interest, DNA of interest, or target DNA) from a DNA extract (DNA template)

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Requirements for PCR

  • DNA Extract

  • Polymerase (Thermus aquaticus)

  • Primers

  • 4 dNTPs

  • Buffer Solution

  • Thermal Cycler

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Basic Principles of PCR

Denature, Anneal, Extend

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Denature

  • separation of two strands of DNA

  • carried out at a temperature of 94°C

  • double-stranded DNA is denatured into single-stranded DNA (H-bonds denature)

<ul><li><p>separation of two strands of DNA </p></li><li><p>carried out at a temperature of <strong>94°C </strong></p></li><li><p>double-stranded DNA is denatured into single-stranded DNA (H-bonds denature)</p></li></ul><p></p>
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Anneal

  • primer hybridization temperature

  • carried out at a temperature generally between and 40 and 70°C

  • uses Primers

<ul><li><p>primer hybridization temperature </p></li><li><p>carried out at a temperature generally between and 40 and 70°C </p></li><li><p>uses Primers</p></li></ul><p></p>
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Primers (Anneal)

short, single-stranded sequences complementary to regions that flank the DNA to be amplified

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Extends

  • carried out at a temperature of 72°C

  • synthesis of the complementary strand

  • Polymerase binds to the primed single-stranded DNAs and catalyze replication using the dNTPS present in the reaction mixture

<ul><li><p>carried out at a temperature of 72°C </p></li><li><p>synthesis of the complementary strand </p></li><li><p>Polymerase binds to the primed single-stranded DNAs and catalyze replication using the dNTPS present in the reaction mixture</p></li></ul><p></p>
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Agarose Gel Electrophoresis

a procedure that reveals stained DNA via ultraviolet transillumination (280-320 nm)

<p>a procedure that reveals stained DNA via ultraviolet transillumination (280-320 nm)</p>
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Analysis and Techniques Based on PCR

  1. Microsatellites

  2. Single nucleotide polymorphism (SNPs)

  3. Amplification of fragment length polymorphism (AFLP)

  4. Restriction fragment length polymorphism (RFLP)

  5. Mitochondrial DNA polymorphism (mtDNA)

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Variations of PCR

  1. Reverse transcriptase PCR (RT-PCR)

  2. Quantitative PCR in real time (quantitative real-time PCR)

  3. Semi-quantitative or competitive PCR

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DNA Sequencing Technologies

First Generation, 2nd Generation, 3rd Generation

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1st Gen Sequencing

  • Maxam-Gilbert Sequencing

  • Sanger Sequencing

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2nd Gen Sequencing

  • 454 Pyrosequencing

  • Illumina Sequencing

  • SOLiD Sequencing

  • Ion Torrent Sequencing

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3rd Gen Sequencing

  • PacBio RS System

  • Nanopore Sequencing Technology

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1st Gen | Maxam-Gilbert Sequencing

sequencing by chemical degradation requiring chemical modifications of the DNA and further cleavage and electrophoresis

<p>sequencing by chemical degradation requiring chemical modifications of the DNA and further cleavage and electrophoresis </p>
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[STEPS] Maxam-Gilbert

  1. Radioactive labeling of the 5’-P ends of dsDNA with 32P-dATP

  2. Denature with DMSO at 90°C then electrophoresis

  3. Modification of the nitrogenous bases

  4. Chemical cleavage of the ssDNA at the 5’-P side

  5. Electrophoresis and autoradiography

  6. Infer DNA sequence

<ol><li><p>Radioactive labeling of the 5’-P ends of dsDNA with <sup>32</sup>P-dATP</p></li><li><p>Denature with DMSO at 90°C then electrophoresis </p></li><li><p>Modification of the nitrogenous bases</p></li><li><p>Chemical cleavage of the ssDNA at the 5’-P side </p></li><li><p>Electrophoresis and autoradiography </p></li><li><p>Infer DNA sequence</p></li></ol><p></p>
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[DISADVANTAGES] Maxam-Gilbert

  • Uses hazardous chemicals

  • Technically challenging

  • Difficult to scale-up

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1st Gen | Sanger Sequencing

a nucleic-acid sequencing approach by enzymatic synthesis, aka chain termination method

<p>a nucleic-acid sequencing approach by enzymatic synthesis, aka chain termination method</p>
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[STEPS] Sanger Sequencing

  1. Denature the dsDNA

  2. Anneal with primers

  3. Extend with dNTPS and ddNTPs

  4. Segregation via gel or capillary electrophoresis

  5. Interpret virtual electropherogram or electrofluorogram

<ol><li><p>Denature the dsDNA </p></li><li><p>Anneal with primers </p></li><li><p>Extend with dNTPS and ddNTPs</p></li><li><p>Segregation via gel or capillary electrophoresis</p></li><li><p>Interpret virtual electropherogram or electrofluorogram</p></li></ol><p></p>
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[DISADVANTAGES] Sanger Sequencing

  • Expensive (equipment, reagents, workforce)

  • Time consuming

  • Not suitable for genomic sequencing

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2nd Gen | Roche 454 Pyrosequencing

emulsion-PCR technology generating the longest reads (from 200 to 1000b)

<p>emulsion-PCR technology generating the longest reads (from 200 to 1000b) </p>
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[STEPS] Roche 454 Pyrosequencing

  1. Library construction = nebulization, adapter ligation, biotin-tagged DNA attaches to streptavidin in beads

  2. Emulsion PCR

  3. DNA-enriched beads to picotiter wells + smaller beads containing enzymes (ATP sulfurylase, luciferase, and apyrase)

  4. Single nucleotide is flown, signal detection (base calling), wash/degrade remaining bases

  5. Sequencing = image/signal processing

<ol><li><p>Library construction = nebulization, adapter ligation, biotin-tagged DNA attaches to streptavidin in beads </p></li><li><p>Emulsion PCR </p></li><li><p>DNA-enriched beads to picotiter wells + smaller beads containing enzymes (ATP sulfurylase, luciferase, and apyrase)</p></li><li><p>Single nucleotide is flown, signal detection (base calling), wash/degrade remaining bases </p></li><li><p>Sequencing = image/signal processing </p></li></ol><p></p>
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[DISADVANTAGES] Roche 454 Pyrosequencing

  • Homopolymeric regions —> InDel errors

  • Challenging sample prep

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2nd Gen | Illumina Sequencing

  • Reversible-terminator sequencers generating shorter reads (35-150b) but larger genome coverages

<ul><li><p>Reversible-terminator sequencers generating shorter reads (35-150b) but larger genome coverages</p><p></p></li></ul><p></p>
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[STEPS] Illumina Sequencing

  1. Library construction = fragmentation, ligation of “Y” adapters, immobilization

  2. Cluster generation = PCR or bridge amplification

  3. Sequencing = reversible-terminator sequencing using fluorescent nucleotides

  4. Data Analysis = assemble the reads into contigs

<ol><li><p>Library construction = fragmentation, ligation of “Y” adapters, immobilization </p></li><li><p>Cluster generation = PCR or bridge amplification </p></li><li><p><strong>Sequencing = reversible-terminator sequencing using fluorescent nucleotides</strong></p></li><li><p>Data Analysis = assemble the reads into contigs </p></li></ol><p></p>
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[DISADVANTAGES] Illumina Sequencing

  • Substitution errors

  • Signal decay (lower accuracy at ends)

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2nd Gen | SOLiD Sequencing

Sequencing by Oligonucleotide Ligation and Detection (SOLiD) can also generate a high coverage, yet with short reds (25-75 bases)

<p>Sequencing by Oligonucleotide Ligation and Detection (SOLiD) can also generate a high coverage, yet with short reds (25-75 bases)</p>
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[STEPS] SOLiD Sequencing

  1. Library Construction —> emPCR —> DNA immobilized in glass surface

  2. Sequencing = five universal sequencing primers (with 2 interrogative bases) —> ligate —> fluorescent probes = repeated in n-1, n-2, n-3, & n-4

  3. Data Analysis = assemble the reads

<ol><li><p>Library Construction —&gt; emPCR —&gt; DNA immobilized in glass surface </p></li><li><p>Sequencing = five universal sequencing primers (with 2 interrogative bases) —&gt; ligate —&gt; fluorescent probes = repeated in n-1, n-2, n-3, &amp; n-4</p></li><li><p>Data Analysis = assemble the reads </p></li></ol><p></p>
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[DISADVANTAGES] SOLiD Sequencing

  • Highly complex

  • Challenging sample prep

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2nd Gen | Ion Torrent Sequencing

based on the detection of the protons generated on the polymerization reactions of nucleic acids

<p>based on the detection of the protons generated on the polymerization reactions of nucleic acids</p>
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[STEPS] Ion Torrent Sequencing

  1. Library construction —> emPCR —> DNA immobilized in picowells

  2. Sequencing = one - by - one dNTP-flush extension detection the proton released

  3. Data Analysis = assemble the reads

<ol><li><p>Library construction —&gt; emPCR —&gt; DNA immobilized in picowells </p></li><li><p>Sequencing = one - by - one dNTP-flush extension detection the proton released </p></li><li><p>Data Analysis = assemble the reads</p></li></ol><p></p>
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[DISADVANTAGES] Ion Torrent Sequencing

  • Still relies on clonally amplified template

  • Errors in homopolymeric regions

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3rd Gen | PacBio SMRT Sequencing

Single-molecule real-time (SMRT) sequencing using special loop adapters and zero mode wavelength design (ZMW)

<p>Single-molecule real-time (SMRT) sequencing using special loop adapters and zero mode wavelength design (ZMW)</p>
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[STEPS] PacBio SMRT Sequencing

  1. Library construction = follows strand displacement amplification (rolling circle)

  2. Sequencing = fluorescently labeled base (P) —> light emission

  3. Data Analysis = assemble the reads

<ol><li><p>Library construction = follows strand displacement amplification (rolling circle) </p></li><li><p>Sequencing = fluorescently labeled base (P) —&gt; light emission </p></li><li><p>Data Analysis = assemble the reads </p></li></ol><p></p>
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[DISADVANTAGES] PacBio SMRT Sequencing

  • Very high error rates

  • Requires high molecular weight template

  • Costly

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3rd Gen | Nanopore Sequencing

based on the detection of the nitrogenous bases as they move through pores embedded in a lipid bilayer on microwells

<p>based on the detection of the nitrogenous bases as they move through pores embedded in a lipid bilayer on microwells </p>