Page 28 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

322 Chapter 9 Digital Analysis of DNA

These variables are the strength of the electric field applied and agarose (formed by the noncovalent association of
across the gel, the composition of the gel, the charge per unit agarose polymers), which is suitable for looking at larger
volume of the DNA molecule (known as charge density), and fragments up to about 20 kb as in Fig. 9.3.
the physical size of the molecule. The only one of these varia-
bles that actually differs among any set of linear DNA frag- essential concepts
ments migrating in a particular gel is size. The reason is that
all the DNA molecules are subjected to the same electric field • Restriction enzymes cut DNA molecules at specific
and the same gel matrix, and they all have the same charge sequences; mechanical shearing breaks DNA at
density (because the charge of all nucleotide pairs is nearly random locations.
identical). As a result, only differences in size cause different • The longer the sequence recognized by a restriction
linear DNA molecules to migrate at different speeds during enzyme, the fewer but larger will be the fragments the
electrophoresis. enzyme produces when cutting genomic DNA.
The longer a molecule of linear DNA, the larger the • Certain restriction enzymes can produce fragments that
volume it occupies as a random coil. The larger the vol- all have the same sticky ends.
ume a molecule occupies, the less likely it is to find a pore • Gel electrophoresis separates DNA fragments according
in the gel matrix big enough to squeeze through, and the to their sizes. The smaller the fragment, the farther it will
more often it will bump into the matrix. And the more migrate in the gel.
often the molecule bumps into the matrix, the lower its
rate of migration (also referred to as its mobility). Thus, in
any given period of electrophoresis, smaller DNAs will
travel greater distances from the wells than larger DNAs. 9.2 Cloning DNA Fragments
When electrophoresis is completed, the gel is incubated
with a fluorescent DNA-binding dye called ethidium bro-
mide. After the unbound dye has been washed away, it is learning objectives
easy to visualize the DNA by placing the gel under an ultra-
violet light, which causes the dye bound to the DNA frag- 1. Diagram the process by which restriction enzymes and
ments to glow in an orange color. You can determine the DNA ligase are used to make recombinant DNA
actual sizes of DNA molecules on gels by comparing their molecules.
migration distances to those of known marker fragments 2. Describe how scientists produce cellular clones of
subjected to electrophoresis in an adjacent lane of the gel. recombinant DNA molecules.
Figure 9.3 (step 5) illustrates the types of results ob- 3. Contrast the use of plasmid vectors with that of BAC or
tained by analyzing different DNA samples. If a genome is YAC (bacterial or yeast artificial chromosome) vectors.
small, such as the 48.5 kb constituting the chromosome of 4. Explain why genomic DNA libraries require more colonies
the bacterial virus bacteriophage lambda (λ), then EcoRI than are contained by a single genome equivalent.
restriction enzyme digestion of this DNA will produce a
small number of discrete bands that can be discriminated
from each other easily by gel electrophoresis and whose The smear of hundreds of thousands of different DNA frag-
sizes total 48.5 kb. In contrast, electrophoresis of the hun- ments seen in the gel of EcoRI-cut human DNA in Fig. 9.3
dreds of thousands of different fragments created when a suggests that the genomes of animals, plants, and even mi-
sample of human genomic DNA is treated with the same croorganisms like E. coli are so complex that you can make
enzyme will generate a smear centered around the average sense of them only by looking at a small piece at a time.
fragment size (about 4.1 kb for EcoRI, as previously dis- Ideally, you would like to purify just one of these fragments—a
cussed). Random breakage of DNA by mechanical forces tiny bit of the genome—away from all the other fragments.
will also produce a distribution of fragments whose aver- You would then like to amplify this particular DNA fragment—
age size will reflect the intensity of the shearing forces ap- that is, make many identical DNA copies of it. Amplification
plied to the sample (not shown). would allow you to obtain enough DNA to study, the most
DNA molecules range in size from small fragments of obvious type of analysis being to determine the sequence of
fewer than 10 bp to whole human chromosomes that have nucleotides that constitute this particular fragment. If you
an average length of 130,000,000 bp. No one sizing proce- could sequence separately each of the hundreds of thousands
dure has the capacity to separate molecules throughout this of fragments, you might ultimately be able to figure out the
enormous range. To detect DNA molecules in different size genome’s entire DNA sequence.
ranges, researchers use a variety of protocols based mainly The process that uses living cells both to isolate a sin-
on two kinds of gels: polyacrylamide (formed by covalent gle fragment of DNA from a complex mixture and to make
bonding between acrylamide monomers), which is good many exact replicas of that fragment is called molecular
for distinguishing smaller DNA fragments (less than 1 kb); cloning. This technique was central to the initial success of