Medical Laboratory Science Harr - 8.1
Molecular Diagnostics: Molecular
Methods Exam
Which double-stranded DNA molecule has the highest melting temperature?
A. An oligonucleotide with a repeating sequence of A-A-A at the 5´ end
B. A molecule of 5,000 base pairs with a high number of A-T base pairs
C. An oligonucleotide with a large number of repeating C-G-C codons
D. A DNA polymer of 100,000 base pairs - Answers -C. An oligonucleotide with a large
number of repeating C-G-C codons
C 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 dsDNA to ssDNA. This is done by breaking the hydrogen bonds between
base pairs. A-T base pairs have two hydrogen bonds, while 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.
Which base pair sequence is most likely to serve as a binding site for a restriction
endonuclease?
A. A-T-T-C-A
T-A-A-G-T
B. C-T-A-C-T-G
G-A-T-G-A-C
C. C-A-C
G-T-G
D. A-A-G-C-T-T
T-T-C-G-A-A - Answers -D. A-A-G-C-T-T
T-T-C-G-A-A
D Restriction endonucleases are enzymes that cut double-stranded DNA 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 upon 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.
Cloning a human gene into a bacterium in order to make a large molecular probe
requires which vector?
A. Plasmid
B. Bacterial microsome
C. 30S bacterial ribosome
D. Single-stranded DNA - Answers -A. Plasmid
A A plasmid is a piece of circular double-stranded DNA located in the cytoplasm of a
bacterium. Although not attached to a chromosome, the plasmid is replicated like
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.
What process can be used to make a DNA probe produce a fluorescent or
chemiluminescent signal?
A. Enzymatic attachment of acridinium esters to terminal ends of the probe
B. Substitution of biotinylated or fluorescent nucleotides into the probe
C. Splicing the gene for β-galactosidase into the probe
D. Heat denaturation of the probe followed by acid treatment - Answers -B. Substitution
of biotinylated or fluorescent nucleotides into the probe
B 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 (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.
What term describes the products produced when DNA is digested by restriction
endonucleases?
A. Mosaicisms
B. Chimeras
C. Amplicons
D. Restriction fragment length polymorphisms - Answers -D. Restriction fragment length
polymorphisms
D 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 the polymerase chain reaction (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 RFLPs for 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, such as when contamination occurs repeatedly
or when the genes to be analyzed comprise a length of DNA too long for efficient
amplification.
The following figure shows a DNA size standard (ladder) made by restriction enzyme
digestion (PstI) of lambda phage DNA that has been separated by agarose gel
electrophoresis. Which DNA band has the highest molecular weight?
A. 1
B. 2
C. 3
D. 4 - Answers -A. 1
A Each phosphoric acid subunit within a phosphodiester bond to adjacent deoxyribose
molecules has a single negative charge at an alkaline pH. Since 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
What reagent is most commonly used to stain DNA separated by electrophoresis?
Molecular Diagnostics: Molecular
Methods Exam
Which double-stranded DNA molecule has the highest melting temperature?
A. An oligonucleotide with a repeating sequence of A-A-A at the 5´ end
B. A molecule of 5,000 base pairs with a high number of A-T base pairs
C. An oligonucleotide with a large number of repeating C-G-C codons
D. A DNA polymer of 100,000 base pairs - Answers -C. An oligonucleotide with a large
number of repeating C-G-C codons
C 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 dsDNA to ssDNA. This is done by breaking the hydrogen bonds between
base pairs. A-T base pairs have two hydrogen bonds, while 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.
Which base pair sequence is most likely to serve as a binding site for a restriction
endonuclease?
A. A-T-T-C-A
T-A-A-G-T
B. C-T-A-C-T-G
G-A-T-G-A-C
C. C-A-C
G-T-G
D. A-A-G-C-T-T
T-T-C-G-A-A - Answers -D. A-A-G-C-T-T
T-T-C-G-A-A
D Restriction endonucleases are enzymes that cut double-stranded DNA 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 upon 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.
Cloning a human gene into a bacterium in order to make a large molecular probe
requires which vector?
A. Plasmid
B. Bacterial microsome
C. 30S bacterial ribosome
D. Single-stranded DNA - Answers -A. Plasmid
A A plasmid is a piece of circular double-stranded DNA located in the cytoplasm of a
bacterium. Although not attached to a chromosome, the plasmid is replicated like
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.
What process can be used to make a DNA probe produce a fluorescent or
chemiluminescent signal?
A. Enzymatic attachment of acridinium esters to terminal ends of the probe
B. Substitution of biotinylated or fluorescent nucleotides into the probe
C. Splicing the gene for β-galactosidase into the probe
D. Heat denaturation of the probe followed by acid treatment - Answers -B. Substitution
of biotinylated or fluorescent nucleotides into the probe
B 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 (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.
What term describes the products produced when DNA is digested by restriction
endonucleases?
A. Mosaicisms
B. Chimeras
C. Amplicons
D. Restriction fragment length polymorphisms - Answers -D. Restriction fragment length
polymorphisms
D 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 the polymerase chain reaction (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 RFLPs for 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, such as when contamination occurs repeatedly
or when the genes to be analyzed comprise a length of DNA too long for efficient
amplification.
The following figure shows a DNA size standard (ladder) made by restriction enzyme
digestion (PstI) of lambda phage DNA that has been separated by agarose gel
electrophoresis. Which DNA band has the highest molecular weight?
A. 1
B. 2
C. 3
D. 4 - Answers -A. 1
A Each phosphoric acid subunit within a phosphodiester bond to adjacent deoxyribose
molecules has a single negative charge at an alkaline pH. Since 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
What reagent is most commonly used to stain DNA separated by electrophoresis?
