Файл: Cell and Molecular Biology [High-Yield].pdf

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MOLECULAR BIOLOGY TECHNIQUES

Electrophoresis:

• When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides), the gel is

usually composed of different concentrations of acrylamide and a cross-linker, producing differ-
ent sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater than
a few hundred bases), the preferred matrix is purified agarose.

• After a DNA sample is cut into fragments by an RE, the DNA fragments can be separated from

one another by PAGE based on size of the DNA fragments.

• The sizes of the DNA fragments can be compared and a physical map (called a restriction map)

of the DNA sample can be constructed showing the location of each cut site.

• A DNA sample is cut with either EcoR1 or HindIII REs.
• The mixture of DNA fragments obtained from the RE treatment is placed at the top of the agarose

gel slab and under an electric field, the DNA fragments move through the gel toward the posi-
tive electrode because DNA is negatively charged.

• Smaller DNA fragments migrate faster than large DNA fragments and thus the DNA fragments

in the mixture become separated by size.

• Note that smaller DNA fragments are located at the bottom of the gel and  larger DNA frag-

ments are located at the top of the gel.

• To visualize the DNA fragments in the gel, the gel is soaked in a dye that binds to DNA and flu-

oresces under ultraviolet light.

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CHAPTER 14

A

B

● Figure 14-3

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MOLECULAR BIOLOGY TECHNIQUES

The Enzymatic Method of DNA Sequencing.

Although restriction maps provide useful

information concerning a DNA sample, the ultimate physical map of DNA is its nucleotide
sequence. The nucleotide sequence is established by a technique called DNA sequencing. This
method employs the use of DNA synthesis with dideoxyribonucleoside triphosphates which lack
the 3

-OH group that is normally found on deoxyribonucleoside triphosphates. If a dideoxyri-

bonucleoside triphosphate becomes incorporated into DNA during synthesis, the addition of the
next nucleotide is blocked due to the lack of the 3

-OH group. This forms the basis of the enzy-

matic method of DNA sequencing.

Figure 14-3A The biochemical structure of deoxyribonucleoside triphosphates (dGTP, dATP,

dTTP, dCTP) and dideoxyribonucleoside triphosphates (ddGTP, ddATP, ddTTP, ddCTP) is shown.
Note the lack of the 3

-OH group on the dideoxyribonucleoside triphosphates.

Figure 14-3B:

• Double-stranded DNA is separated into single strands and one of the strands is used as the tem-

plate.

• A radiolabeled primer (ATGC) is used to initiate DNA synthesis.
• Four separate reaction mixtures are set up containing DNA polymerase, dGTP, dATP, dTTP,

dCTP and ddGTP, ddATP, ddTTP, or ddCTP. These four reactions will produce a number of dif-
ferent length DNA fragments which will terminate in G, A, T, or C depending on what dideoxyri-
bonucleoside triphosphate was present in the reaction mixture.

• The contents of each reaction mixture are separated by gel electrophoresis based on size of the

DNA fragments.

• The gel is then exposed to film such that the radiolabeled primer will identify each of the DNA

fragments as bands. The bands are arranged as four parallel columns representing DNA frag-
ments of varying lengths that terminate in G, A, T, or C.

• A typical DNA sequencing film is shown and you may be asked on the USMLE to read a sequenc-

ing gel.

• Start at the bottom the film and identify the lowest band (i.e., the shortest DNA fragment) and

note that the lowest band in found in the T column (S). Now you know that the first nucleotide
in the sequence is T.

• Go the next lowest band on the film and note that it is found under the G column (S). Now

you know that the second nucleotide in the sequence is G.

• Continue this process for all 26 bands.
• Note that when you start at the bottom of the film and go up, you will be constructing the DNA

sequence in a 5

 S 3 direction.

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CHAPTER 14

Nitrocellulose

membane

P

32

-radiolabeled

DNA probe

A

B

● Figure 14-4

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MOLECULAR BIOLOGY TECHNIQUES

Southern Blotting and Prenatal Testing for Sickle Cell Anemia.

Southern blotting

allows for the identification of a specific DNA sequence (e.g., gene for the 

-globin chain of hemo-

globin) by using a DNA probe and the hybridization reaction. A DNA probe is a single-stranded
piece of DNA (10–120 base pairs oligonucleotide) that participates in a hybridization reaction. A
hybridization reaction is a reaction whereby a single-stranded piece of DNA (like a DNA probe)
binds (or hybridizes) with another piece of single-stranded DNA of complementary nucleotide
sequence. The hybridization reaction exploits a fundamental property of DNA to denature and
renature. The two strands of double-helix DNA are held together by weak hydrogen bonds that
can be broken (denatured) by high temperature (90

C) or alkaline pH such that single-stranded

DNA is formed. Under low temperature or acid pH, single-stranded DNA will reform double-helix
DNA (renature). A Southern blot is used to detect major gene rearrangements and deletions found
in a variety of human diseases. A Southern blot can also be used to identify structurally related
genes in the same species and homologous genes in other species. Basically, a Southern blot gives
information whether a gene is present or absent but does not give information about the expres-
sion of the gene.

Figure 14-4A Southern blotting:

• Double-stranded DNA is cut by three different REs and separated by gel electrophoresis in three

separate lanes. One lane is reserved for radiolabeled DNA size markers.

• The double-stranded DNA is transferred to a nitrocellulose membrane under alkaline conditions

so the DNA is denatured into single strands.

• The nitrocellulose paper is placed in a plastic bag along with the radiolabeled P

32

DNA probe

and incubated under conditions that favor hybridization.

• The nitrocellulose paper is exposed to photographic film (autoradiography) so that the radiola-

beled probe will show up as bands.

Figure 14-4B Prenatal testing for sickle cell anemia. It is good news when you hear that a gene

has been cloned and sequenced because now a DNA probe that hybridizes to the gene can be made
and used, for example, in prenatal testing for sickle cell anemia. Sickle cell anemia is a recessive
genetic disease caused by a mutation in the 

-globin gene that results in a change of single amino

acid from glutamic acid (normal) to valine (mutant) in the 

-globin protein. Both the normal gene

and mutant gene for 

-globin protein have been sequenced so that DNA probes can be made to

locate both of these genes in a Southern blot.

• Fetal DNA (F) is obtained from a high-risk fetus and compared with control DNA (C1 & C2).

The DNA is separated into two samples. Each sample is cut with REs, subjected to gel elec-
trophoresis, and transferred to nitrocellulose paper under denaturing conditions.

• One sample is hybridized with a DNA probe for the normal 

-globin gene and the other sample

is hybridized with a DNA probe for the mutant 

-globin gene.

• After autoradiography, the films A and B can be analyzed.
• You will likely be asked to interpret a Southern blot on the USMLE for an autosomal recessive,

autosomal dominant, or X-linked genetic disease.

• Examine lane F (fetal DNA) in films A and B. Note that lane F has no bands in film A (no nor-

mal 

-globin gene) but one band in film B (mutant -globin gene). This means that the fetus is

homozygous for the mutant 

-globin gene and therefore will have sickle cell anemia.

• Examine lane C1 (control DNA) in films A and B. Note that lane C1 has one band in film A but

no bands in film B. This means that this person is homozygous for the normal 

-globin gene and

therefore will be normal.

• Examine lane C2 (control DNA) in films A and B. Note that lane C2 has one band in film A and

one band in film B. This means that this person is heterozygous having one copy of the normal
-globin gene and one copy of the mutant -globin gene. This person will be normal because
sickle cell anemia is genetic recessive disease so that two copies of the mutant 

-globin gene are

necessary for sickle cell anemia to appear.

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