Development of DNA Sequencing Technologies

Question 1

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

Answer 1

Molecular Biology

Question 2

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

Answer 2

DNA Replication

Question 3

DNA Replication follows these 3 steps

Answer 3

Unwind
Prime
Elongate

Question 4

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

Answer 4

^E DNA Polymerase

Question 5

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

Answer 5

^U DNA Helicase

Question 6

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

Answer 6

^U Single-Stranded DNA Binding Proteins (SSBs)

Question 7

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

Answer 7

^U Topoisomerase

Question 8

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

Answer 8

^P Primase

Question 9

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

Answer 9

^E DNA Ligase

Question 10

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).

Answer 10

Polymerase Chain Reaction

Question 11

Requirements for PCR

Answer 11

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

Question 12

3 Basic Principles of PCR

Answer 12

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

Question 13

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

Answer 13

Primers

Question 14

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

Answer 14

Agarose Gel Electrophoresis (AGE)

Question 15

Analysis and Techniques Based on PCR

Answer 15

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)

Question 16

Variations of PCR

Answer 16

1. Reverse transcriptase PCR (RT-PCR)
2. Quantitative PCR in real time (quantitative real-time PCR)
3. Semi-quantitative or competitive PCR

Question 17

DNA Sequencing Technologies

Answer 17

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

Question 18

First Generation Sequencing Technologies

Answer 18

o Maxam-Gilbert Sequencing
o Sanger Sequencing

Question 19

Second Generation Sequencing Technologies

Answer 19

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

Question 19

Third Generation Sequencing Technologies

Answer 19

o PacBio RS System
o Nanopore Sequencing Technology

Question 20

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

Answer 20

1st Gen: Maxam-Gilbert Sequencing

Question 21

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

Question 22

Disadvantages of 1st Gen: Maxam-Gilbert Sequencing:

Answer 22

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

Question 23

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

Answer 23

1st Gen: Sanger Sequencing

Question 24

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

Question 25

Disadvantages of 1st Gen: Sanger Sequencing

Answer 25

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

Question 26

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

Answer 26

2nd Gen: Roch 454 Pyrosequencing

Question 27

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

Question 28

Disadvantages of 2nd Gen: Roch 454 Pyrosequencing

Answer 28

❑Homopolymeric regions → InDel errors
❑Challenging sample prep

Question 29

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

Answer 29

2nd Gen: Illumina Sequencing

Question 30

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

Question 31

Disadvantages of 2nd Gen: Illumina Sequencing

Answer 31

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

Question 32

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

Answer 32

2nd Gen: SOLiD Sequencing

Question 33

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

Question 34

Disadvantages of 2nd Gen: SOLiD Sequencing

Answer 34

❑Highly complex
❑Challenging sample prep

Question 35

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

Answer 35

2nd Gen: Ion Torrent Sequencing

Question 36

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

Question 37

Disadvantages of 2nd Gen: Ion Torrent Sequencing

Answer 37

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

Question 38

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

Answer 38

3rd Gen: PacBio SMRT Sequencing

Question 39

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

Question 40

Disadvantages of 3rd Gen: PacBio SMRT Sequencing

Answer 40

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

Question 41

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

Answer 41

3rd Gen: Nanopore Sequencing