CUTT1INGDN4

Question 1

Cells manipulate DNA by using

______ proteins that catalyze specific chemical reactions

Answer 1

enzymes:

Question 2

Molecular biologists get enzymes from ____, then use the

enzymes in new ways.

Answer 2

cells

Question 3

_______ cut DNA at specific sites. They recognize short sequences of nucleotides, called _______, and cut the DNA at those sites.

Answer 3

Restriction enzymes

restriction sites

Question 4

Restriction sites are usually _______ base pairs long

Answer 4

four to eight

Question 5

________ are also called endonucleases, because they cut nucleic acids somewhere in the midst of the molecule (endo- means in).

Answer 5

Restriction enzymes

Question 6

________ were the first DNA altering enzymes to be isolated and used in the laboratory; in a sense, molecular biology began when a restriction enzyme was used to
cut DNA in a test tube

Answer 6

Restriction enzymes

Question 7

Restriction enzymes cut up DNA. Wouldn’t this be a bad thing for a cell? Not if it’s DNA that can harm the cell. The restriction enzymes you’ll use come from ______,
and the _____ use the enzymes to cut up DNA from viruses or other potentially dangerous sources

Answer 7

bacteria

Question 8

_______ function as a self-defense weapon for the cell.

Answer 8

restriction enzymes

Question 9

How do the enzymes
avoid cutting up the
cell’s own DNA?

Answer 9

A bacterial cell may lack restriction sites that can be cut by its own
enzymes, or it may chemically modify the DNA (by adding methyl
groups) at the restriction sites

Question 10

WHY IS CUTTING DNA USEFUL IN THE LAB?

One key reason for cutting DNA in the lab is ____________. That’s the essence of gene cloning.

Answer 10

so you can join different DNA pieces together through ligation

Question 11

Two different restriction enzymes to cut one kind of DNA:

______ - Isolated from Escherichia coli.

______- Isolated from Haemophilus
influenzae.

Answer 11

Eco R1

Hin DIII

Question 12

The DNA is from a virus. The virus is called ___; it’s a bacteriophage, meaning it’s a virus that infects bacteria.

When you use electrophoresis to look at your results (next lab period), you’ll be able to see
the difference from one reaction to another, because the enzymes cut the lambda DNA into different-sized fragments. The uncut DNA
should give _____ on the gel, while the cut DNAs will show various _______.

Answer 12

lambda
one big band
smaller bands

Question 13

Enzymes are proteins, and they depend on having the right 3-dimensional shape to do their job. Since the shape of a protein is controlled in part by weak interactions such as______, it can be altered by changes in ________ When you buy an enzyme, it comes with a buffer that ensures the right environment for the enzyme. The
buffer comes in a separate tube, usually as a 10x concentrate. You dilute it down to 1x before
adding the enzyme.

Answer 13

hydrogen bonds

pH or salt concentration.

Question 13

______is one of the most commonly used techniques in molecular biology.

Answer 13

Electrophoresis

Question 14

In DNA electrophoresis, pieces of DNA are separated from one another on the _________.

Answer 14

basis of their size

Question 15

DNA in solution is a _____molecule. Remember, DNA is deoxyribonucleic acid, and like all acids, it can give up a proton in solution. Once it gives up the proton, the DNA has a ______. You can use the charge to make the molecule move.

Answer 15

charged

negative charge

Question 16

In electrophoresis, you put the DNA samples into a ______ and apply an _______ through the solution. Electric currents have a positive end and a negative end, and salty water conducts electricity well. The electric current creates an electric field,
similar to the field around a magnet. DNA, being a ________, migrates toward the positive pole of the
electric current or electric field.

Answer 16

salty solution
electric current
negatively charged molecule

Question 17

The trick with electrophoresis is to get ____________s to migrate toward the ______ at different rates. You can accomplish this by putting the DNA in an _______.

Answer 17

different-sized DNA molecules
positive pole
agarose gel

Question 18

An _____ is like Jell-O –

it’s a solid matrix, consisting largely of complex carbohydrates, and filled with water.

Answer 18

agarose gel

Question 19

______comes from the manufacturer as a powder; when you boil it in water, it turns into a gel.

Answer 19

Agarose

Question 20

Electrophoresis . .

All the DNA molecules move in the ____ direction, but their rate of migration depends on how ___ they are. The small DNA
molecules can _____through the pores in the agarose, so they move rapidly; the larger
pieces are _____ because they don’t fit through the pores as well.

Answer 20

same
big
easily fit
slowed down

Question 21

The speed at which molecules move through gels is controlled by:

The size of the molecule (smaller = \_\_\_\_).
The charge of the molecule (uncharged won’t migrate; stronger charge = \_\_\_).
The voltage applied to the electrophoresis unit (more voltage = \_\_\_).
The density (or percent agarose) of the gel (\_\_\_\_\_ % agarose = faster).
The type of agarose (some are denser than others).

Answer 21

faster
faster
faster
lower

Question 22

In a single DNA gel, the difference in the rates at which various DNA bands move is all due to ___. All the DNA in one gel has the same charge and is subject to the same ____and ____.

Answer 22

size
gel density
voltage

Question 23

_______ are DNA fragments

of known size that are used to determine the size of unknown DNA.

Answer 23

Molecular weight markers

Question 24

_______ are often made by taking

some DNA and cutting it into specific pieces using a restriction enzyme.

Answer 24

Molecular weight markers

Question 25

DNA comes in all sizes, and you need to match the gel to the size of the DNA you want to look at. A _____ has small pores, and
large DNA molecules won’t easily fit through those pores. For large DNA molecules, you
need a ______

Answer 25

dense gel

less dense gel.

Question 26

______is measured in terms of % agarose. A gel with 0.5 g agarose dissolved in 50 ml buffer is a 1% gel.

Answer 26

Gel density

Question 27

DNA in solution is ____. It doesn’t reflect visible light. In order for you to learn anything from your gel, you need to use a trick to
visualize the DNA.

Answer 27

invisible

Question 28

common reagent for visualizing DNA:

Answer 28

ethidium bromide.

Question 29

_______ is a dye

that binds specifically to DNA.

Answer 29

Ethidium bromide

Question 30

Ethidium bromide is a dye that binds specifically to DNA. When the ethidium is bound to the DNA, it becomes ______. If you shine an ultraviolet light on it, the DNA-ethidium complex absorbs the UV and emits ______ visible light. This allows researchers to see DNA in gels, even in very small quantities.

Answer 30

highly fluorescent

bright orange

Question 31

______is handy, but it’s also one of the more hazardous chemicals you’ll use this
quarter.

Answer 31

Ethidium bromide

Question 32

Ethidium bromide is a ______. Ethidium binds to DNA, in a gel or in your cells. Like most
things that bind to DNA, ethidium can alter the conformation of the DNA. This can lead to errors
when the cell copies the DNA. Hence, ethidium is a mutagen, which means that it causes
mutations, or changes in the nucleotide sequence of DNA. Your DNA is just fine the way it is. You
don’t want to mutate it.

Answer 32

mutagen

Question 33

Mutagens are also often ______; by causing changes in DNA, they
increase the risk of cancer. Ethidium should be considered a potential carcinogen, though it has not
been proven carcinogenic.

Answer 33

carcinogens

Question 34

The power supplies for electrophoresis supply a fairly _______ to the buffer
in the electrophoresis chamber (although at a fairly low current). The chamber is designed so that
current can only flow through it when the lid is on. When the lid is on, you won’t be able to stick
your fingers in the buffer chamber. This eliminates most of the potential shock hazard.

Answer 34

high voltage

Question 35

Hot agarose in the microwave: you’ll need to boil your agarose in its buffer to prepare the
gel. Hot agarose can boil over very easily, and could burn you. Only ____ should go in the microwave at a time to reduce the risk of spills. Use a _____ to protect your hand.

Answer 35

one flask

rubber flask gripper

Question 35

Ultraviolet light: you’ll be using a _________UV light to illuminate your gel. UV light can be very bad for your eyes. Fortunately, we have a device that virtually eliminates the possibility of exposing yourself to this light. The device is a _______ so called because it shines the light through the gel. The transilluminator has a lid on it that blocks all the UV
but allows the visible light to come through.

Answer 35

high-intensity, short-wavelength

UV transilluminator,

Question 36

DNA samples need to be mixed with a _____ before loading them on a gel.

Answer 36

loading buffer

Question 37

The loading buffer does three things: it ___ the DNA sample so you can see it when you load it on the gel, it
makes the sample ___ enough to sink to the bottom of the well, and it contains a dye that
migrates with the DNA so you can tell how far it has gone.

Answer 37

colors

dense

Question 38

The gel and the gel buffer contain ______ and must be treated as hazardous waste.

Answer 38

ethidium bromide,

Question 39

READING THE GEL

The DNA that you loaded in one well will run in one ___. Each lane may contain one or more _____.

Answer 39

lane

bands

Question 40

Note that the largest DNA fragments are at the______, closest to the well;
they migrate slowest.

Also note that the highest molecular weight bands are the ____; you
can see them better because there’s more DNA there.

Answer 40

top

brightest

Question 41

Larger pieces of DNA need to be run in gels with a ___ percentage of agarose and at____ voltages. The
conditions for running the gel need to match the size of the DNA you’re looking for.

Answer 41

lower

Question 42

One lane of your gel should have a ________ such as the lambda/Hind III shown
above. The other lanes will have your ______ samples along with uncut lambda DNA.

Answer 42

molecular weight marker

digest and ligation

Question 43

If you know the sizes of the bands in one lane, you can determine the size of a band in another lane. If your unknown band ran at exactly the same speed as the 4,361-bp band of lambda/Hind III, then it’s the same size. If your unknown band is in between two bands, you can ___.

Answer 43

interpolate

Question 44

Most people usually just guess at the sizes of their bands, after comparing with the appropriate
molecular weight marker. However, you could get out a____ and measure if you want to be
more precise. If you ___ the distance each band migrated, you’ll find that the rate of migration is proportional to the log of the fragment size in base pairs. If you plot it out on ______
paper, you’ll get a straight line. Then it will be easy to determine the exact size of your unknown
band. However, you don’t need to do that today.

Answer 44

ruler
graph
semilog graph