Molecular Flashcards

Question

Answer 1

A

C. An oligonucleotide with a large number of repeating C-G-C codons

The melting temperature of DNA refers to the temperature required to separate the
molecule into single strands. The Tm is the temperature required to convert half of the
DNA from double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA). This is
done by breaking the hydrogen bonds between base pairs. A-T base pairs have two
hydrogen bonds, whereas C-G base pairs have three. Therefore, molecules with a high
proportion of C-G base pairs are more resistant to heat denaturation or melting.

Answer 2

A

D. A-A-G-C-T-T T-T-C-G-A-A

Restriction endonucleases are enzymes that cut dsDNA into fragments and are
important tools used in molecular diagnostics. Each restriction enzyme recognizes a specific oligonucleotide sequence, and the size and number of fragments it produces when DNA is digested depend on the number of times that sequence is repeated in the DNA molecule. Restriction endonucleases recognize palindromic sequences (i.e., the base sequence of complementary strands reads the same from opposite directions). The
sequence
A-A-G-C-T-T
T-T-C-G-A-A
is the recognition site for HindIII, a restriction endonuclease isolated from Haemophilus influenzae. If a disease gene produces a base pair substitution at the restriction site, the enzyme will not recognize it and not cut the DNA. This results in a longer fragment that can be recognized by electrophoresis. This process was initially used to identify the hemoglobin S gene using the restriction enzyme MstII. The point mutation changes an A to a T within the restriction site, causing loss of the normal-sized fragment.

Answer 3

A

A. Plasmid

A plasmid is a piece of circular dsDNA located in the cytoplasm of a bacterium.
Although not attached to a chromosome, the plasmid is replicated similar to
chromosomal DNA. The plasmid is cut with the restriction endonuclease that is used to
isolate the DNA fragment containing the gene of interest. The fragment anneals to the sticky ends of the plasmid DNA, and the cut is repaired by DNA ligase. The
recombinant plasmid is added to a culture of bacteria that is disrupted to promote the
uptake of plasmid DNA. Commercially available plasmids have promoter and reporter genes, such as lac and lacZ, that produce β-galactosidase. These can be used to identify colonies with successful recombinants. They also carry antibiotic resistance genes that allow the recombinants to be purified. Culture of the recombinant bacteria results in large amounts of the gene, which can be harvested using the restriction enzyme,
denatured, and labeled to make the probe.

Answer 4

A

B. Substitution of biotinylated or fluorescent nucleotides into the probe

Fluorescent or enzyme labels can be attached to probes by nick translation. A DNase is used to cut the probe at a few phosphodiester linkages. PolI repairs the nicks by removing nucleotides from the 3’ end and replacing them with labeled nucleotides at the 5’ end of the nick. Alternatively, a primer containing a labeled nucleotide can be used to make copies of the probe by DNA amplification (polymerase chain reaction [PCR]). A common label used for probes consists of biotin conjugated to the 5’ end of the probe. After hybridization, streptavidin conjugated to an enzyme, such as alkaline phosphatase, is added. Streptavidin strongly binds to biotin, forming an enzyme-labeled complex with the DNA. After washing to remove unbound streptavidin, a colorimetric, fluorescent, or chemiluminescent substrate is added.

Answer 5

A

D. Restriction fragment length polymorphisms

Mosaicism occurs when cells within the same individual contain different numbers of chromosomes and results from nondisjunction during early embryonic development. Chimeras are molecules created when translocation occurs between genes (exons) on different chromosomes. Amplicons are exact copies of a DNA template produced by DNA amplification techniques, such as PCR. When a restriction enzyme cuts two different DNA molecules, the size of some fragments will differ because the number and position of restriction sites differ. Such fragments are called restriction fragment length polymorphisms (RFLPs). Analysis of RFLPs can be used to test for disease genes, study genetic linkage, and establish identity. It is used usually when PCR is impractical, as when contamination occurs repeatedly or when the genes to be analyzed comprise a length of DNA too long for efficient amplification.

Answer 6

A

A. 1
Each phosphoric acid subunit within a phosphodiester bond to adjacent deoxyribose molecules has a single negative charge at an alkaline pH. Because the charge is distributed evenly, smaller fragments move more rapidly through the gel. When
suspended in an alkaline buffer (pH 8), such as tris-borate-EDTA (TBE) or tris-acetate-
EDTA (TAE), the DNA fragments migrate toward the anode at a rate that is inversely
proportional to the log10 of molecular size. If the distance traveled is plotted against the
log of molecular weight, the plot will be a straight line with a negative slope because
the larger the molecule, the more slowly it moves through the pores of the gel. The plot
can be calibrated with a DNA size ladder, and the molecular weight of DNA fragments
can be determined from the calibration curve.

Answer 7

A

D. Ethidium bromide

When ethidium bromide inserts between the base pairs of dsDNA, the dye becomes
fluorescent, releasing 480 nm light when stimulated by long wavelength ultraviolet
(UV) light. Ethidium bromide staining has a sensitivity of approximately 10 ng/mL
(1.5 ng per band) DNA. It is frequently added to molten agarose or capillary
electrophoresis (CE) buffer at a concentration of 0.5 μg/mL to visualize and quantify DNA. Its binding to ssDNA and RNA is not as efficient as that of more sensitive dyes, such as SYBR gold, picoGreen, and YOYO-1.

Answer 8

A

A. Southern blot

Southern blot hybridization is a method commonly used to detect disease genes in both PCR products and RFLP testing. The DNA fragments are electrophoresed, and the
DNA bands are transferred by suction to a nylon or nitrocellulose membrane. The
bands are immobilized and denatured on the membrane, and a solution containing the labeled probe is added. Hybridization is the binding of the complementary base
sequence of the probe to the target sequence. This process is highly dependent on temperature, ionic strength, and the presence of reagents in the hybridization solution that influence stringency (the degree of exactness of base pairing). A Northern blot test follows the same process, except that the sample is RNA. In a Western blot test, the sample is a mixture of proteins, and the probes used are (labeled) antibodies to the proteins of interest. A dot blot is a hybridization method in which samples of DNA are placed directly on the nitrocellulose membrane as a circular spot (or bar in the case of a slot blot), followed by the hybridization process.

Molecular Flashcards

(90 cards)