Lecture 16 - Methods of nucleic acid analysis

Question 1

DNA sequencing determines

Answer 1

the exact sequence of a DNA molecule

Question 2

In solid-phase synthesis of nucleic acids,

Answer 2

sequences of nucleic acids can be synthesized and used to identify or amplify other nucleic acids

Question 3

Restriction enzymes

Answer 3

“cut” precise DNA segments allowing for manipulation of a given sequence

Question 4

Blotting techniques are used to

Answer 4

separate and characterize DNA (southern) and RNA (western)

Question 5

Fluorescent in situ hybridization (FISH)

Answer 5

locates DNA/RNA sequences in the cell

Question 6

The polymerase chain reaction

Answer 6

amplifies a segment of DNA

Question 7

Microarrays

Answer 7

quantify the expression of genes

Question 8

RNAseq

Answer 8

quantifies the copy number of each RNA molecule in a sample

Question 9

Sanger sequencing is based on

Answer 9

generation of DNA fragments whose length depends on the last base in the sequence

Question 10

In Sanger sequencing, collections of DNA fragments can be generated by

Answer 10

controlled termination of replication (Sanger dideoxy method)

Question 11

The four reaction mixtures in Sanger sequencing all contain

Answer 11

• DNA polymerase
• DNA primer
• Radiolabeled dATP, TTP, dGTP, dCTP
• 2’, 3’-dideoxy analog

Question 12

In Sanger sequencing, a chemically synthesized primer is required for the DNA polymerase to

Answer 12

start synthesizing the complementary strand

Question 13

In Sanger sequencing, most of the new growing DNA molecules consist of

Answer 13

one of the four deoxyribonucleosides (they’re in excess)

Question 14

In Sanger sequencing, occasionally, a

Answer 14

dideoxy analog will be incorporated into the growing chain (blocks further growth)

Question 15

Why isn’t Sanger sequencing used for sequencing the entire genome?

Answer 15

Not fast enough

Question 16

Developing automatic DNA sequencers based on ___________ allowed more rapid sequencing of larger amounts of DNA

Answer 16

fluorescent dideoxynucleotide chain terminators

Question 17

Pseudogenes

Answer 17

resemble functional genes but are no longer expressed

Question 18

Pseudogenes are formerly functional genes that

Answer 18

picked up mutations

Question 19

Pseudogenes are transcribed into

Answer 19

RNA and function to regulate parental genes

Question 20

Solid-phase DNA synthesis can

Answer 20

immobilize the growing product while flushing soluble contaminants and by-products

Question 21

Steps in solid-phase DNA synthesis

Answer 21

1. 3’phosphorus atom of incoming nucleotide is joined to 5’ oxygen fo growing chain
2. Phosphate triester is oxidized by iodine
3. Removal of DMT protecting group on 5’-OH of growing chain
4. NH3 is added to remove remaining protecting groups

Question 22

Restriction enzymes recognize

Answer 22

specific sequences in dsDNA and cleave the DNA at a specific place

Question 23

Restriction enzymes can be used to

Answer 23

• Analyze chromosome structure
• Sequence long DNA molecules
• Isolate genes
• Create new DNA molecules

Question 24

Restriction enzymes cleave by

Answer 24

hydrolyzing a phosphodiester bond in each strand of DNA

Question 25

Cleavage sites in restriction enzymes are often

Answer 25

palindromic

Question 26

Restriction enzymes selectively degrade

Answer 26

foreign DNA but not their own DNA

Question 27

Restriction enzymes may cut DNA resulting in

Answer 27

1. sticky ends 2. blunt ends

Question 28

A specific gene or fragment of DNA may be cleaved several times, creating a

Answer 28

pattern of restriction fragments that are unique

Question 29

Gel electrophoresis separates molecules based on

Answer 29

size and charge

Question 30

What charge does DNA have?

Answer 30

negative

Question 31

In gel electrophoresis, DNA is usually stained with

Answer 31

ethidium bromide

Question 32

Radioactive DNA can be detected by

Answer 32

autoradiography

Question 33

Polyacrylamide gels are used to

Answer 33

separate fragments containing up to 1000 bp

Question 34

Agarose gels are used to

Answer 34

resolve mixtures of larger fragments (up to 20 kb)

Question 35

Which is more porous, polyacrylamide gel or agarose gel?

Answer 35

Agarose gel

Question 36

A Southern blot is based on

Answer 36

hybridization of the DNA fragment of interest with a labeled complementary DNA strand

Question 37

Type of blots used with Southern blotting

Answer 37

DNA

Question 38

Type of blots used with Northern blotting

Answer 38

RNA

Question 39

Type of blots used with Western blotting

Answer 39

Protein

Question 40

Fluorescent in situ hybridization (FISH) is a

Answer 40

cytogenetic technique

Question 41

FISH uses

Answer 41

fluorescent probes that bind to the parts of a chromosome with a high sequence complementarity

Question 42

FISH can be used in identifying specific features in

Answer 42

DNA

Question 43

3 steps of PCR

Answer 43

1. Strand separation 2. Hybridization of primers 3. DNA synthesis

Question 44

In PCR, the two strands of DNA are denatured by

Answer 44

heating

Question 45

In PCR, the primers are hybridized by

Answer 45

cooling the solution

Question 46

In PCR, the solution is heated to _______ for DNA synthesis

Answer 46

72 degrees Celsius

Question 47

What is mixed in PCR?

Answer 47

1. DNA to be amplified (template) 2. DNA polymerase (Taq) 3. primers 4. dNTPs

Question 48

A great advantage of PCR is the

Answer 48

exponential amplification of DNA molecules

Question 49

Quantitative PCR (qPCR) is used to investigate

Answer 49

mRNA levels

Question 50

The fluorescent reporter molecule used in qPCR reactions is typically

Answer 50

SYBR Green

Question 51

In probe-based qPCR, a fluorescent reporter dye is attached to

Answer 51

the 5' end of a probe

Question 52

In probe-based qPCR, a quencher is attached to the

Answer 52

3' end of a probe

Question 53

RNA-seq (RNA sequencing) uses next-generation sequencing (NGS) to

Answer 53

reveal the presence and quantity of RNA in a biological sample at a given moment in time