Development of DNA Sequencing Technologies Flashcards

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1
Q

is 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

A

Molecular Biology

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2
Q

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

A

DNA Replication

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3
Q

DNA Replication follows these 3 steps

A

Unwind
Prime
Elongate

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4
Q

The enzymes responsible for constructing new DNA strands during replication or DNA repair. It can only elongate DNA when there is a free 3’-OH group that it can act on

A

^E DNA Polymerase

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5
Q

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

A

^U DNA Helicase

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6
Q

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

A

^U Single-Stranded DNA Binding Proteins (SSBs)

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7
Q

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

A

^U Topoisomerase

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8
Q

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

A

^P Primase

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9
Q

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

A

^E DNA Ligase

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10
Q

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).

A

Polymerase Chain Reaction

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11
Q

Requirements for PCR

A

✓ DNA Extract
✓ Polymerase (Taq)
✓ Primers
✓4 dNTPs
✓ Buffer Solution
✓ Thermal Cycler

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12
Q

3 Basic Principles of PCR

A

Denature
✓separation of the two strands of DNA
✓carried out at a temperature of 94°C
✓ double-stranded DNA is denatured into single-stranded DNA

Anneal
✓primer hybridization temperature
✓carried out at a temperature generally between 40 and 70°C

Extend
✓carried out at a temperature of 72°C
✓synthesis of the complementary strand
✓Polymerase binds to primed single-stranded DNAs and catalyzes replication using the dNTPS present in the reaction mixture

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13
Q

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

A

Primers

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14
Q

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

A

Agarose Gel Electrophoresis (AGE)

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15
Q

Analysis and Techniques Based on PCR

A
  1. Microsatellites
  2. Single nucleotide polymorphisms (SNPs)
  3. Amplification of fragment length polymorphism (AFLP)
  4. Restriction fragment length polymorphism (RFLP)
  5. Mitochondrial DNA polymorphisms (mtDNA)
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16
Q

Variations of PCR

A
  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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17
Q

DNA Sequencing Technologies

A

First Generation Sequencing
o Maxam-Gilbert Sequencing
o Sanger Sequencing

Second Generation Sequencing
o 454 Sequencing
o Illumina Sequencing
o SOLiD Sequencing
o Ion Torrent Sequencing

Third Generation Sequencing
o PacBio RS System
o Nanopore Sequencing Technology

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18
Q

First Generation Sequencing Technologies

A

o Maxam-Gilbert Sequencing
o Sanger Sequencing

19
Q

Second Generation Sequencing Technologies

A

o 454 Sequencing
o Illumina Sequencing
o SOLiD Sequencing
o Ion Torrent Sequencing

19
Q

Third Generation Sequencing Technologies

A

o PacBio RS System
o Nanopore Sequencing Technology

20
Q

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

A

1st Gen: Maxam-Gilbert Sequencing

21
Q

1st Gen: Maxam-Gilbert Sequencing

Steps:
❑Radioactive labeling of the 5’-P ends of dsDNA with 32P-dATP
❑Denature with DMSO at 90°C then electrophoresis
❑Modifications of the nitrogenous bases
❑Chemical cleavage of the ssDNA at the 5’-P side
❑Electrophoresis and autoradiography
❑Infer DNA sequence

A
22
Q

Disadvantages of 1st Gen: Maxam-Gilbert Sequencing:

A

❑Uses hazardous chemicals
❑Technically challenging
❑Difficult to scale-up

23
Q

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

A

1st Gen: Sanger Sequencing

24
Q

1st Gen: Sanger Sequencing

Steps:
❑Denature the dsDNA
❑Anneal with primers
❑Extend with dNTPs and ddNTPs
❑Segregation via gel or capillary electrophoresis
❑Interpret virtual electropherogram or electrofluorogram

A
25
Q

Disadvantages of 1st Gen: Sanger Sequencing

A

❑Expensive (equipment, reagents, workforce)
❑Time consuming
❑Not suitable for genomic sequencing

26
Q

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

A

2nd Gen: Roch 454 Pyrosequencing

27
Q

2nd Gen: Roch 454 Pyrosequencing

Steps:
❑Library construction = nebulization, adapter ligation, biotin-tagged DNA attaches to streptavidin in beads
❑Emulsion PCR
❑DNA-enriched beads to picotiter wells + smaller beads containing enzymes (ATP sulfurylase, luciferase, and apyrase) ❑Single nucleotide is flown, signal detection (base calling), wash/degrade remaining bases
❑Sequencing = Image/signal processing

A
28
Q

Disadvantages of 2nd Gen: Roch 454 Pyrosequencing

A

❑Homopolymeric regions → InDel errors
❑Challenging sample prep

29
Q

❑reversible-terminator sequencers generating shorter reads (35-150b) but larger genome coverages

A

2nd Gen: Illumina Sequencing

30
Q

2nd Gen: Illumina Sequencing
Steps:
❑Library construction = fragmentation, ligation of “Y” adapters, immobilization
❑Cluster generation = PCR or bridge amplification
❑Sequencing = reversible-terminator sequencing using fluorescent nucleotides
❑Data Analysis = assemble the reads into contigs

A
31
Q

Disadvantages of 2nd Gen: Illumina Sequencing

A

❑Substitution errors
❑Signal decay (lower accuracy at ends)

32
Q

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

A

2nd Gen: SOLiD Sequencing

33
Q

2nd Gen: SOLiD Sequencing
Steps:
❑Library construction → emPCR → DNA immobilized in glass surface
❑Sequencing = five universal sequencing primers (with 2 interrogating bases) → ligate → fluorescent probes = repeated in n-1, n-2, n-3, & n-4
❑Data Analysis = assemble the reads

A
34
Q

Disadvantages of 2nd Gen: SOLiD Sequencing

A

❑Highly complex
❑Challenging sample prep

35
Q

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

A

2nd Gen: Ion Torrent Sequencing

36
Q

2nd Gen: Ion Torrent Sequencing
Steps:
❑Library construction → emPCR → DNA immobilized in picowells
❑Sequencing = one-by-one dNTP-flush extension detecting the proton released
❑Data Analysis = assemble the reads

A
37
Q

Disadvantages of 2nd Gen: Ion Torrent Sequencing

A

❑Still relies on clonally amplified template
❑Errors in homopolymeric regions

38
Q

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

A

3rd Gen: PacBio SMRT Sequencing

39
Q

3rd Gen: PacBio SMRT Sequencing
Steps:
❑Library construction = follows strand displacement amplification (rolling circle)
❑Sequencing = fluorescently labeled base (P) → light emission
❑Data Analysis = assemble the reads

A
40
Q

Disadvantages of 3rd Gen: PacBio SMRT Sequencing

A

❑Very high error rates
❑Requires high molecular weight template
❑Costly

41
Q

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

A

3rd Gen: Nanopore Sequencing

41
Q
A