Molecular Tools for Studying Genes and Gene Activity P1

Question 1

It is very often necessary in
molecular biology research to separate _______ or _________ from each other.

Answer 1

proteins or nucleic acids

Question 2

For example, we may need to purify a particular enzyme from a ________ extract in order to use it or to study its properties.

Answer 2

crude cellular

Question 3

_________ uses a gel as an
anticonvective medium or sieving
medium during electrophoresis, the
movement of a charged particle in an
electric current.

Answer 3

Gel electrophoresis

Question 4

(PICTURE 1 REFER TO PPT slide 4)

This is a horizontal gel made of agarose. The agarose melts at _______, then gels as it cools. A “______” is inserted into the molten agarose; after the gel cools, the comb is removed, leaving slots, or wells. The DNA is then placed in the wells, and an electric current is run through the gel. Because the DNA is an acid, it is ______charged at neutral pH and electrophoreses, or
migrates, toward the positive pole, or anode.

Answer 4

high temperature ; comb ; negatively

Question 5

(PICTURE 1 REFER TO PPT slide 4)

A photograph of a gel after electrophoresis showing the DNA fragments as bright bands. DNA binds to a _____ that fluoresces orange under ultraviolet light, but the bands appear pink in this photograph.

Answer 5

dye

Question 6

(Determining the Size of a Large DNA by Gel Electrophoresis)

1. The relationship between the log of a DNA’s size and its electrophoretic mobility deviates _____ from linearity if the DNA is _____

Answer 6

strongly ; very large.

Question 7

(Determining the Size of a Large DNA by Gel Electrophoresis)

2. Double-stranded DNA is a relatively rigid rod—very long and thin. The longer it is, the more fragile it is. In fact, _____ DNAs break very easily; even seemingly mild manipulations, like swirling in a beaker or pipetting, create shearing forces sufficient to fracture them.

Answer 7

large

Question 8

A kind of gel electrophoresis that can separate DNA molecules up to several million base pairs (megabases, Mb) long and maintain a relatively linear relationship between the log of their sizes and their mobilities.

Answer 8

(Pulsed-Field Gel Electrophoresis)

Question 9

Instead of a constant current through the gel, this method uses pulses of current, with relatively long pulses in the forward direction and shorter pulses in the opposite, or even sideways, direction.

Answer 9

(Pulsed-Field Gel Electrophoresis)

Question 10

Is valuable for measuring the sizes of DNAs even as large as some of the chromosomes found in yeast

Answer 10

(Pulsed-Field Gel Electrophoresis)

Question 11

(Polyacrylamide Gel Electrophoresis)

Electrophoresis is also often applied to ______, in which case the gel is usually made of _______.

Answer 11

proteins ; polyacrylamide

Question 12

To determine the polypeptide makeup of a complex protein, the experimenter must treat the protein so that the polypeptides, or subunits, will electrophorese independently.

Answer 12

(Polyacrylamide Gel Electrophoresis)

Question 13

(Polyacrylamide Gel Electrophoresis)

This is usually done by treating the protein with a detergent (________ or _____) to denature the subunits so they no longer bind to one another.

Answer 13

sodium dodecyl sulfate, or SDS)

Question 14

(SDS Advantages)

1. It coats all the ______ with negative charges, so they all _______ toward the anode.

Answer 14

polypeptides ; electrophorese

Question 15

(SDS Advantages)

2. It masks the natural charges of the subunits, so they all electrophorese according to their molecular masses and not by their native charges. _______fit easily through the pores in the gel, so they migrate rapidly. ________ migrate more slowly.

Answer 15

Small polypeptides ; Larger polypeptides

Question 16

(Two-Dimensional Gel Electrophoresis)

(1)

The mixture of proteins is electrophoresed through a narrow tube gel containing molecules called ______ that set up a pH gradient from one end of the tube to the other.

A negatively charged molecule will electrophorese toward the _____ until it reaches its isoelectric point, the pH at which it has no net charge.

Without net charge, it is no longer drawn toward the anode, or the cathode, for that matter, so it stops. This step is called ________ because it focuses proteins at their isoelectric points in the gel.

Answer 16

ampholytes ; anode ; isoelectric focusing

Question 17

(Two-Dimensional Gel Electrophoresis)

(2)

The gel is removed from the tube and placed at the top of a slab gel for ordinary ________. Now the proteins that have been partially resolved by _______ are further resolved according to their sizes by SDS-PAGE.

Answer 17

SDS-PAGE ; isoelectric focusing

Question 18

The investigators grew E. coli cells in the presence or absence of ________. Then they stained a lysate of the cells grown in the absence of benzoic acid with the red fluorescent dye _____, so the proteins from that lysate would fluoresce red. They stained a lysate of the cells grown in the presence of benzoic acid with the blue fluorescent dye ____, so those proteins would fluoresce blue.

Answer 18

benzoic acid ; Cy3 ; Cy5

Question 19

They performed two- dimensional gel electrophoresis on:

Answer 19

1. The proteins from cells grown in the absence of benzoic acid
2. On the proteins grown in the presence of benzoic acid.
3. The proteins that accumulate only in the absence of benzoic acid fluoresce red, those that accumulate only in the presence of benzoic acid fluoresce blue, and those that accumulate under both conditions fluoresce both red and blue, and so appear purple or black.

Question 20

(Ion-Exchange Chromatography)

1. Ion-exchange chromatography uses _____ to separate substances, including proteins, according to their charges.

Answer 20

resin

Question 21

(Ion-Exchange Chromatography)

2. Positively charged resins like _______ are used for anion-exchange chromatography, and negatively charged resins like ___________ are used for cation-exchange chromatography.

Answer 21

DEAE-Sephadex ; phosphocellulose

Question 22

(Gel Filtration Chromatography)

1. __________ is one method that separates molecules based on their physical dimensions.

Answer 22

Gel filtration chromatography

Question 23

(Gel Filtration Chromatography)

2. Gel filtration resins such as ______ are porous beads of various sizes that can be likened to “whiffle balls, ” hollow plastic balls with holes in them.

Answer 23

Sephadex

Question 24

3. ________ uses columns filled with porous resins that let in smaller substances, but exclude larger ones. Thus, the smaller substances are slowed in their journey through the column, but larger substances travel relatively rapidly through the column.

Answer 24

Gel filtration chromatography

Question 25

(Affinity Chromatography)

1. The ______ contains a substance (an affinity reagent) to which the molecule of interest has strong and specific affinity.

Answer 25

resin

Question 26

(Affinity Chromatography)

2. The power of affinity chromatography lies in the specificity of binding between the ______ on the resin and the _______ to be purified.

Answer 26

affinity reagent ; molecule

Question 27

(Affinity Chromatography)

3. The molecule of interest binds to a _____ coupled to the affinity reagent but all or most other molecules flow through without binding. Then the molecule of interest can be _____ from the column with a solution of a substance that disrupts the specific binding.

Answer 27

column ; eluted

Question 28

Labeled Tracers

Answer 28

Autoradiography
Phosphorimaging
Liquid Scintillation Counting
Nonradioactive Tracers

Question 29

is a means of detecting radioactive compounds with a photographic emulsion. The form of emulsion favored by molecular biologists is a piece of x-ray film.

Answer 29

Autoradiography

Question 30

(Autoradiography)

2. To enhance the sensitivity of autoradiography, at least with 32P, one can use an __________. This is a screen coated with a compound that fluoresces when it is excited by ________ at low temperature.

Answer 30

intensifying screen ; b electrons

Question 31

(Autoradiography)

3. To measure the exact amount of radioactivity in a fragment of DNA, one can get a rough estimate by looking at the ______ of a band on an autoradiograph, and an even better estimate by scanning the autoradiograph with a __________.

Answer 31

intensity ; densitometer

Question 32

(__________)

1. It is much more accurate in quantifying the amount of _________ in a substance. This is because its response to radioactivity is far more linear than that of an x-ray film.

Answer 32

Phosphorimaging ; radioactivity

Question 33

False color phosphorimager scan of an RNA blot.

After hybridizing a radioactive probe to an RNA blot and washing away unhybridized probe, the blot was exposed to a ___________ plate. The plate collected energy from β electrons from the radioactive probe bound to the RNA bands, then gave up this energy when scanned with a laser. A computer converted this energy into an image in which the colors correspond to radiation intensity according to the following color scale: ——–

Answer 33

phosphorimager

yellow (lowest) , purple, magenta, light blue, green, dark blue, black (highest).

Question 34

Liquid scintillation counter:

1. Uses the __________ from a sample to create photons of visible light that a photomultiplier tube can detect.

Answer 34

radioactive emissions

Question 35

(Liquid Scintillation Counting)

2. __________ is an instrument that lowers the vial into a dark chamber with a photomultiplier tube.

Answer 35

Liquid scintillation counter

Question 36

(Nonradioactive Tracers)

1. A ________ can compete with the sensitivity of a radioactive by using the _________ of an enzyme. An enzyme is coupled to a probe that detects the molecule of interest, so the enzyme will produce many molecules of product, thus amplifying the signal.

Answer 36

nonradioactive tracer ; multiplier effect

Question 37

(Nonradioactive Tracers)

2. To avoid the expense of a phosphorimager or x-ray film, one can use ________ that change color instead of becoming chemiluminescent. These chromogenic substrates produce colored bands corresponding to the location of the enzyme and, therefore, to the location of the molecule of interest.

Answer 37

enzyme substrates

Question 38

(Southern Blots: Identifying Specific DNA Fragments)

1. Begins by using a _________ to cut genomic DNA isolated from the organism. It is best to use a restriction enzyme such as _____ or ______ that recognizes a 6-bp cutting site.

Answer 38

restriction enzyme ; EcoRI or HindIII

Question 39

(Southern Blots: Identifying Specific DNA Fragments)

2. _________ was the pioneer of this technique; he transferred, or blotted, DNA fragments from an agarose gel to nitrocellulose by diffusion

Answer 39

Edward Southern

Question 40

(Southern Blots: Identifying Specific DNA Fragments)

3. ____________ can be used to hybridize to DNAs of the same, or very similar, sequence on a Southern blot. The number of bands that hybridize to a short probe gives an estimate of the number of closely related genes in an organism.

Answer 40

Labeled DNA (or RNA) probes

Question 41

(Southern Blotting)

First, _______ DNA fragments in an agarose gel.

Next, ______ the DNA with base and transfer the single-stranded DNA fragments from the gel (yellow) to a sheet of nitrocellulose or another DNA- binding material (red)

Answer 41

electrophorese ; denature

Question 42

(Southern Blotting)

One can do this in two ways:

Answer 42

1. Diffusion
2. Electrophoresis

Question 43

Next, ______ the blot to a labeled probe and detect the labeled bands by
_________ or __________

Answer 43

hybridize ; autoradiography or phosphorimaging.

Question 44

(DNA Fingerprinting and DNA Typing)

1. ___________ in 1985 were investigating a DNA fragment from the gene for a human blood protein, a-globin, when they discovered that this fragment contained a sequence of bases repeated several times. This kind of repeated DNA is called a ___________

Answer 44

Alec Jeffreys and his colleagues ; minisatellite

Question 45

(DNA Fingerprinting and DNA Typing)

2. Because individuals differ in the pattern of repeats of the basic sequence. These patterns are like fingerprints; indeed, they are called ____________

Answer 45

DNA fingerprints.

Question 46

(DNA Fingerprinting and DNA Typing)

3. A DNA fingerprint is really just a Southern blot. To make one, investigators first cut the DNA under study with a restriction enzyme such as _______

Answer 46

HaeIII

Question 47

(DNA Fingerprinting)

A. First, cut the DNA with a ______. In this case, the enzyme HaeIII cuts the DNA in seven places (short arrows), generating eight fragments. Only three of these fragments (labeled A, B, and C according to size) contain the minisatellites, represented by blue boxes. The other fragments (yellow) contain unrelated DNA sequences.

Answer 47

restriction enzyme

Question 48

(DNA Fingerprinting)

B. _______________ the fragments from part (a), which separates them according to their ____. All eight fragments are present in the electrophoresis gel, but they remain invisible. The positions of all the fragments, including the three (A, B, and C) with minisatellites are indicated by dotted lines.

Answer 48

Electrophorese ; sizes

Question 49

(DNA Fingerprinting)

C. _______ the DNA fragments and _________ them

Answer 49

Denature ; Southern blot

Question 50

(DNA Fingerprinting)

D. ______ the DNA fragments on the Southern blot to a labeled DNA with several copies of the minisatellite. This probe will bind to the three fragments containing the minisatellites, but with no others. Finally, use _______ or ________ to detect the three labeled bands.

Answer 50

Hybridize ; x-ray film or phosphorimaging

Question 51

(Forensic Uses of DNA Fingerprinting and DNA Typing)

1. Although almost all individuals have _____ patterns, parts of the pattern (sets of bands) are inherited in a _______. Thus, _______ can be used to establish parentage.

Answer 51

different ; Mendelian fashion ; fingerprints

Question 52

(Forensic Uses of DNA Fingerprinting and DNA Typing)

2. ______ has the potential to identify criminals. This is because a person’s DNA fingerprint is, in principle, unique, just like a traditional fingerprint.

Answer 52

DNA fingerprinting

Question 53

(Forensic Uses of DNA Fingerprinting and DNA Typing)

3. Forensic scientists have developed ______ that hybridize to a single DNA locus that varies from one individual to another, rather than to a whole set of DNA loci as in a ____________

Answer 53

probes ; classical DNA fingerprint

Question 54

(Use of DNA typing to help identify a rapist)

REFER TO SLIDE 25 IN PPT

Two suspects have been accused of attacking and raping a young woman, and DNA analyses have been performed on various samples from the suspects and the woman.

Lanes 1, 5, and 9 contain ________.

Lane 2 contains DNA from the _______ of suspect A.

Lane 3 contains DNA from a _______ sample found on the woman’s clothing.

Lane 4 contains DNA from the blood cells of _______

Lane 6 contains DNA obtained by swabbing the woman’s _______. (Too little of the victim’s own DNA was present to detect.)

Lane 7 contains DNA from the _________.

Lane 8 contains a _______

Lane 10 is a control containing ______. Partly on the basis of this evidence, suspect B was found guilty of the crime.

Note how his DNA fragments in lane 4 match the DNA fragments from the semen in lane 3 and the vaginal swab in lane 6.

Answer 54

marker DNAs ; blood cells ; semen ; suspect B ; vaginal canal ; woman’s blood cells ; control DNA ; no DNA

Question 55

(In Situ Hybridization: Locating Genes in Chromosomes)

1. ________ can also be used to hybridize to chromosomes and thereby reveal which chromosome has the gene of interest. The strategy of such ____________ is to spread the chromosomes from a cell and partially denature the DNA to create single-stranded regions that can hybridize to a labeled probe.

Answer 55

Labeled probes ; in situ hybridization

Question 56

(In Situ Hybridization: Locating Genes in Chromosomes)

2. If the probe is fluorescently labeled, the technique is called ________________.

Answer 56

fluorescence in situ hybridization (FISH)

Question 57

_____________

1. Although they do not use hybridization, follow the same experimental pattern as Southern blots: The investigator electrophoreses molecules and then blots these molecules to a membrane where they can be identified readily. However, immunoblots involve _________ of proteins instead of nucleic acids.

Answer 57

Immunoblots (Western blots) ; electrophoresis

Question 58

Immunoblots (Western blots)

2. Proteins can be detected and quantified in complex mixtures using ___________. Proteins are electrophoresed, then blotted to a membrane and the proteins on the blot are probed with specific antibodies that can be detected with labeled secondary antibodies or protein A.

Answer 58

immunoblots (or Western blots)

Question 59

(The Sanger Chain-Termination Sequencing Method)

1. The Sanger DNA sequencing method uses __________ to terminate DNA synthesis, yielding a series of DNA fragment whose sizes can be measured by electrophoresis.

Answer 59

dideoxy nucleotides

Question 60

(The Sanger Chain-Termination Sequencing Method)

2. The _______ in each of the fragments is known, because we know which dideoxy nucleotide was used to terminate each reaction.

Answer 60

last base

Question 61

(The Sanger Chain-Termination Sequencing Method)

3. Therefore, ordering these fragments by ____, each fragment one base longer than the next, tells us the base sequence of the DNA.

Answer 61

size

Question 62

(Automated DNA Sequencing)

_________ make the process of Sanger DNA sequencing method very efficient.

Answer 62

Automated sequenators

Question 63

(___________)

1. Allows very rapid sequencing of ______ if the genome of one member of the species has already been sequenced.

Answer 63

High-Throughput Sequencing ; genomes

Question 64

(High-Throughput Sequencing)

2. One such high-throughput method, called _____________, has the great advantages of speed and accuracy. In ____________, nucleotides are added one by one, and the incorporation of a nucleotide is detected by the release of pyrophosphate, which leads through a chain of reactions to a flash of light.

Answer 64

pyrosequencing

Question 65

(High-Throughput Sequencing)

3. Another method, developed by the _________, uses short pieces of DNA amplified in tiny, closely spaced patches on a support surface.

Answer 65

Illumina company

Question 66

(Restriction Mapping)

1. A ________ tells us about the spatial arrangement of physical “landmarks”, such as _________, on a DNA molecule

Answer 66

physical map ; restriction sites

Question 67

(Restriction Mapping)

2. One important strategy in restriction mapping (mapping of restriction sites) is to _______ in question with two or more restriction enzymes in separate reactions, measure the sizes of the resulting fragments, then cut each with another restriction enzyme and measure the sizes of the subfragments by _______

Answer 67

cut the DNA ; gel electrophoresis

Question 68

(Restriction Mapping)

3. This strategy reveals overlaps between individual __________.

Answer 68

restriction fragments

Question 69

(Protein Engineering with Cloned Genes)

1. Using _______, we can introduce changes at will, thus altering the amino acid sequences of the protein products.

Answer 69

cloned genes

Question 69

(Protein Engineering with Cloned Genes)

2. The mutagenized DNA can be made with __________, ___________, and ________

Answer 69

double-stranded DNA, two
complementary mutagenic primers, and PCR.

Question 70

(Protein Engineering with Cloned Genes)

3. Simply digesting the PCR product with ______ removes almost all of the wild- type DNA, so cells can be transformed primarily with mutagenized DNA.

Answer 70

DpnI