Chapter 10

Question 1

used routinely for a variety of purposes, including detecting mutations, typing microorganisms, identifying human haplotypes, and designating polymorphisms.

Answer 1

DNA sequence information

Question 2

Ultimately, targeted therapies will be directed at abnormal DNA sequences detected by these techniques

Answer 2

DNA sequence information

Question 3

used to identify exact sequence of nucleotide and bases in DNA.

Answer 3

Lab technique

Question 4

Applications of DNA Sequencing

Answer 4

✓ Genomic research - studying all the DNA of an organism
✓ Personalized Medicine
✓ Cancer diagnosis and treatment
✓ Pharmacogenetics- person’s genes responds to drugs
✓ Forensics
✓ Microbial ecology - relationships of germs w/in microbiomes
✓ Infections diagnosis and tracking
✓ Agricultural biotechnology
✓ Prenatal genetic testing
✓ Comparative genomics - view of how organisms are related
✓ Evolutionary biology - history of life forms on Earth
✓ Population genetics

Question 5

Two types of sequencing methods have been used most extensively:

Answer 5

Maxam-Gilbert method
Sanger method

Question 6

dideoxy chain termination sequencing methods

Answer 6

Dideoxy (Sanger) Sequencing

Question 7

required a single-stranded template.

Answer 7

Dideoxy (Sanger) Sequencing

Question 8

Templates up to a <1,000 few thousand bases long could be produced using

Answer 8

M13 bacteriophage

Question 9

Templates up to a <1,000 few thousand bases long could be produced using M13 bacteriophage

Answer 9

Dideoxy (Sanger) Sequencing

Question 10

is a modification
of the DNA replication process.

Answer 10

Dideoxy chain termination (Sanger) sequencing

Question 11

A short, synthetic single-stranded DNA fragment (primer)
complementary to sequences just 5′ to the region of DNA to be sequenced is used for priming dideoxy sequencing reactions (Fig. 10-4).

Answer 11

Dideoxy (Sanger) Sequencing

Question 12

lack the hydroxyl group found on the 3′ ribose carbon of the deoxynucleotides (dNTPs;

Answer 12

Dideoxynucleotides

Question 13

For sequencing, modified _______________ are added to the reaction mixture.

Answer 13

dideoxynucleotide (ddNTP)

Question 14

DNA Pol

Answer 14

Taq Pol, Klenow Pol

Question 15

Low possestivity
No endonuclease activity

Answer 15

DNA POL

Question 16

Recombinant polymerase enzymes

Answer 16

SEQUENASE

Question 17

✓ high processivity,
✓ no endonuclease activity,
✓ High reaction rate
✓ heat stable enzymes

Answer 17

DNA POL

Question 18

heat stable enzymes

Answer 18

Thermosequence

Question 19

Steps in Sanger Sequencing

Answer 19

1. DENATURATION (94-98C)
2. ANNEALING (50-65C)
3. EXTENSION (72C)
4. TERMINATION

Question 20

If the concentration of ddNTPs is too high, polymerization will terminate ___________ early along the template.

Answer 20

Too frequently

Question 21

If the ddNTP concentration is too low, ___________termination will occur.

Answer 21

infrequent or no

Question 22

chelate cations and stop enzyme activity

Answer 22

20 mM EDTA

Question 23

denature the products of the synthesis reaction

Answer 23

formamide

Question 24

gel loading dyes

Answer 24

bromophenol blue and/or xylene cyanol

Question 25

Depending on the reagents and gel used, the number of bases per sequence read averages

Answer 25

300-400

Question 26

Sequencing reads can also be lengthened by loading the same ladders in intervals of

Answer 26

2-6 hours

Question 27

The fragments migrate through the gel according

Answer 27

Size

Question 28

Too many ddNTPs will result in a

Answer 28

short sequence read.

Question 29

Too low a concentration of ddNTPs will result

Answer 29

loss of sequence data

Question 30

requires a double- or single-stranded version of the DNA region to be sequenced, with one end radioactively labeled.

Answer 30

Chemical (Maxam-Gilbert) Sequencing

Question 31

strong reducing agent of maxam sequencing

Answer 31

10% piperidine

Question 32

The denaturing conditions that can prevent the single strands of DNA from hydrogen bonding with one another or folding up so that they migrate through the gel strictly according to their size

Answer 32

formamide, urea, and heat

Question 33

Can read dsDNA

Answer 33

Next Generation Sequencing

Question 34

Ngs Amplification

Answer 34

Flow cell

Question 35

NGS can be read using

Answer 35

bioinformatics

Question 36

Steps in NGS

Answer 36

1. Ligation = Library preparation 2. Cluster Amplification 3. Sequencing; fluorescent

Question 37

Connect adaptors “synthetic ligase”

Answer 37

Ligation

Question 38

Cluster amplification of NGS USES

Answer 38

Klenow polymerase

Question 39

Perform next-generation sequencing workflow using three basic steps:

Answer 39

library preparation, sequencing, and data analysis.