CUTT1INGDN4 Flashcards
Cells manipulate DNA by using
______ proteins that catalyze specific chemical reactions
enzymes:
Molecular biologists get enzymes from ____, then use the
enzymes in new ways.
cells
_______ cut DNA at specific sites. They recognize short sequences of nucleotides, called _______, and cut the DNA at those sites.
Restriction enzymes
restriction sites
Restriction sites are usually _______ base pairs long
four to eight
________ are also called endonucleases, because they cut nucleic acids somewhere in the midst of the molecule (endo- means in).
Restriction enzymes
________ 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
Restriction enzymes
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
bacteria
_______ function as a self-defense weapon for the cell.
restriction enzymes
How do the enzymes
avoid cutting up the
cell’s own DNA?
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
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.
so you can join different DNA pieces together through ligation
Two different restriction enzymes to cut one kind of DNA:
______ - Isolated from Escherichia coli.
______- Isolated from Haemophilus
influenzae.
Eco R1
Hin DIII
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 _______.
lambda
one big band
smaller bands
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.
hydrogen bonds
pH or salt concentration.
______is one of the most commonly used techniques in molecular biology.
Electrophoresis
In DNA electrophoresis, pieces of DNA are separated from one another on the _________.
basis of their size
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.
charged
negative charge
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.
salty solution
electric current
negatively charged molecule
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 _______.
different-sized DNA molecules
positive pole
agarose gel
An _____ is like Jell-O –
it’s a solid matrix, consisting largely of complex carbohydrates, and filled with water.
agarose gel
______comes from the manufacturer as a powder; when you boil it in water, it turns into a gel.
Agarose
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.
same
big
easily fit
slowed down
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).
faster
faster
faster
lower
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 ____.
size
gel density
voltage
_______ are DNA fragments
of known size that are used to determine the size of unknown DNA.
Molecular weight markers
_______ are often made by taking
some DNA and cutting it into specific pieces using a restriction enzyme.
Molecular weight markers
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 ______
dense gel
less dense gel.
______is measured in terms of % agarose. A gel with 0.5 g agarose dissolved in 50 ml buffer is a 1% gel.
Gel density
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.
invisible
common reagent for visualizing DNA:
ethidium bromide.
_______ is a dye
that binds specifically to DNA.
Ethidium bromide
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.
highly fluorescent
bright orange
______is handy, but it’s also one of the more hazardous chemicals you’ll use this
quarter.
Ethidium bromide
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.
mutagen
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.
carcinogens
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.
high voltage
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.
one flask
rubber flask gripper
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.
high-intensity, short-wavelength
UV transilluminator,
DNA samples need to be mixed with a _____ before loading them on a gel.
loading buffer
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.
colors
dense
The gel and the gel buffer contain ______ and must be treated as hazardous waste.
ethidium bromide,
READING THE GEL
The DNA that you loaded in one well will run in one ___. Each lane may contain one or more _____.
lane
bands
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.
top
brightest
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.
lower
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.
molecular weight marker
digest and ligation
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 ___.
interpolate
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.
ruler
graph
semilog graph
