Chapter 13
Nucleic Acid-Based Methods of Analysis
Deborah T. Newby, Elizabeth M. Marlowe, and Raina M. Maier
1. What are the major advantages of PCR when it is applied to environmental
samples?
 Extremely sensitive, in some cases can detect a single target molecule.
 Culture independent, can detect nucleic acid from viable, nonviable, and viable
but none culturable microbes.
 Fast, analysis can be completed in a few hours compared to several days to weeks
for culture methods.
 PCR fingerprinting techniques may allow discrimination of isolates that would
otherwise not be distinguishable.
 Can evaluate gene expression in the environment using reverse transcriptase PCR.
 Introduces less bias than culturing followed by PCR or other analyses.
2. What are the major disadvantages of PCR when it is applied to environmental
samples?
 PCR does not discern between live and dead cells, simply detects the nucleic acid.
 Sequences must have enough homology with environmental microbes to allow
amplification.
 Conversely, contamination or nonspecific priming can lead to false-positives.
 Nucleic acid extraction efficiency is method and sample dependent.
 Quality of nucleic acids following extraction and purification may influence
ability to conduct PCR due to coextracted impurities such as metals and humic
acids that may inhibit PCR.
3. If you wanted to use PCR to assess microbial activity, what type of PCR would you
use and why?
Reverse transcriptase PCR (RT-PCR) is used to assess microbial activity by analyzing
mRNA or gene transcripts. RT-PCR may be combined with real-time PCR techniques to
provide rapid and quantitative results. The first step is to convert the mRNA to cDNA
using reverse transcriptase. The cDNA is subsequently used as template in standard PCR
sing DNA polymerase to amplify the target sequence.
4. At the beginning of PCR there are two template DNA strands (T). As amplification
proceeds, short strands (S) and long strands (L) are generated. After 5 cycles of
PCR, how many total strands each of T, S, and L are there?
After cycle 1:
After cycle 2:
T=2
S=0
L=2
T=2
S=2
L=4
After cycle 3:
T=2
S=8
L=6
After cycle 4:
T=2
S = 22
L=8
After cycle 5
T=2
S = 52
L = 10
Note that there is a mistake on Page 256 in the table showing equations for the total # of
strands produced. The equation of the number of total strands produced after n cycles should
read:
Total strands produced after n cycles = 2 template + 2n long + {2(n+1) – 2(n+2) + 2} short
5. Compare and contrast ERIC and AP-PCR method of fingerprinting.
ERIC and AP-PCR are both PCR methods used to amplify sequences that may be
repeated at multiple locations throughout the genome of an organism. Analysis of
each method using gel electrophoresis shows multiple bands of various sizes, or what
is referred to as a “molecular fingerprint” for that organism. AP-PCR or arbitrarily
primed-PCR uses a single primer that is 10-20 bp in length and has a random
sequence. ERIC (enterobacterial repetitive intergenic consensus) uses 2 primers with
specific sequences that target inverted repeats. In both cases, conditions such as
DNA quality, PCR temperature profiles can result in non-reproducible fingerprints.
Accordingly, caution should be used for either type of PCR for comparisons made
between different reactions.
6. a. You want to identify, at the genus level, several bacterial strains isolated from
the soil. Explain how you would do this using molecular techniques.
After a bacterial strain has been isolated from the environment it can be identified at
the genus level by using universal 16S rRNA primers to PCR amplify the 16S rRNA
gene of the isolate. Sometimes heating cells releases enough DNA to serve as
template in this reaction, while for some isolates it may be necessary to extract the
DNA from the isolate prior to carrying out the PCR reaction. The resulting 16S
rRNA products can be purified in a via gel electrophoresis and subsequently cut from
the gel, purified further using any number of kits or protocols designed for this
application. The purified PCR products are subsequently sequenced often in a
commercial laboratory. Once the sequence is determined it can be compared with
sequences in a public database such as GenBank (Table 13.1) to determine the closest
match. GenBank contains millions of sequences submitted from researchers around
the world
b. How would you identify a soil bacterium without prior cultivation using
molecular techniques?
A similar approach to that described in the answer to 6a can be used to identify a soil
bacterium without prior cultivation. In this case, community DNA would be
extracted from the soil. PCR amplification using 16S rRNA gene primers would be
used, followed by gel electrophoresis to separate the 16S rRNA amplicons from any
nonspecific amplification products that might have been generated. The 16S rRNA
band would contain many different 16S rRNA sequences since community DNA was
used as template. Cloning can then be used to separate the different sequences from
each other. All the 16S rRNA gene inserts within a single clone population will be
the same. Thus, once you have a clone with a 16S rRNA gene insert, you can conduct
a plasmid prep on the clone, and directly sequence the 16S rRNA insert from the
plasmid.
7. Compare and contrast the lux reporter system and the GFP reporter system. Based
on their characteristics, which would be more useful in evaluating contaminant
transport in a soil column study?




The lux reporter system allows direct collection of the luminescence signal. In contrast,
the GFP reporter signal requires an excitation of the fluorophore followed by signal
collection.
The lux reporter system produces luminescence only while the operon that it is has been
inserted into is expressed. So lux can be used to examine temporal responses of the
operon. In contrast, the GFP is a protein that is produced while the operon is being
expressed and this protein may have a long half life. Thus, GFP is better for instances
where researchers are only interested in whether the operon has been expressed.
The lux reporter system requires both energy and oxygen in order to function. In
contrast, the GFP system does not require cellular metabolism or O2.
The lux reporter system produces only luminescence while the GFP systems have been
constructed to report in different colors (green, red, yellow). Thus, multiple GFP
reporters could be studied in one system.
For reasons mentioned in the above bullets, in terms of a soil column study, the lux reporter
system would have advantages over the GFP (see Figure 13.25). These include no need for
excitation of the signal by an outside light source which would be hard to provide to cells
within a soil column. Second, the signal from a lux reporter system is temporal and
therefore is conducive to monitoring transport of a contaminant.
8. You are trying to characterize isolates that you suspect have the ability to degrade
2,4-D. You isolate two strains (strain 1 and strain 2) and begin to characterize them
using molecular techniques. You screen for the tfd-b gene (one of the genes that
encodes for an enzyme responsible for 2,4-D degradation) using seminested PCR,
generate ERIC PCR fingerprints, and do a plasmid profile to look for the large 80
kb plasmid which carries the 2,4-D genes. Using the well-studied 2,4-D degrader
Ralstonia eutropha as a control, what can you say about each isolate based on the
results shown?
Note the positive control in the figure should read Ralstonia eutropha not Alcaligenes
eutrophis.
From the semi-nested PCR, it appears that both Strain 1 and Strain 2 contains the tfd-b
gene since it co-migrates with the positive control.
From the Eric PCR, it appears that Strain 2 is identical to the positive control.
From the plasmid profile, it appears that both Strain 1 and 2 have an 80kb plasmid.
Conclusion: The data suggest that strain 2 is R. eutropha and that both Strain 1 and
Strain 2 are likely to be able to degrade 2,4-D.
9. The schematic shows the position and orientation of four primers used in nested
PCR. How many amplification products will be obtained?
Only 2 PCR products will be obtained. (No product from left-hand primer on top strand
due to its location relative to the primer on the bottom strand.)
10. There are several features of real-time PCR that make its use advantageous over
conventional PCR. List three and explain why each feature is beneficial.
Examples of acceptable answers:
1) Real-time PCR can be performed in 30 minutes or less compared with 3-4
hours by conventional PCR followed by gel analysis because amplicons are
smaller (shorter extension times), smaller reaction volumes are used, and no postPCR analysis (gel electrophoresis) is needed.
2) Results are analyzed using fluorescence readers built into the real-time PCR
instruments which provides greater sensitivity (down to a single copy) compared
to gel electrophoresis methods.
3) Higher specificity can be achieved using real-time PCR when detection
methods that utilize probes are employed since not only the primers, but the
probe(s) must also bind.
4) Quantification is based on dynamic measurements in the exponential phase of
amplification as compared to gel-based quantifications based on densitometry of
bands in a gel which is an endpoint measurement.
5) Melt analysis can be performed when using dyes such as SYBR Green I which
allows an nearly immediate secondary assessment that the correct product, or at
least one with the expected melting temperature, was generated.
11. Based on Figure 13.18, what was the original concentration of a sample that has a Ct
of 34? Can you determine the concentration for samples with Ct values of 11, 29, 47?
Explain why or why not.
34: You cannot determine the original concentration of a sample that has a Ct of 34
because at that Ct value the blank begins to amplify either due to contamination or
nonspecific amplification. Another acceptable answer would be that this is the Ct of the
blank.
11: cannot determine, outside range of standards, too concentrated
29: cannot determine, outside range of standards, too diluted
47: cannot determine, outside range of standards, too diluted
12. Compare and contrast AFLP and T-RFLP. Which technique would you use to
differentiate closely related isolates and why?
AFLP and T-RFLP are both methods that generate unique banding patterns (DNA
fingerprints) although typically for isolate and community fingerprinting respectively. In
T-RFLP a specific gene is targeted, whereas in AFLP multiple regions of the genome are
amplified and fragmented. There is a higher likelihood that AFLP would allow two
closely related isolates to be differentiated since several different regions (sequences) are
examined versus a single target. It more likely that the two isolates would give the same
band with T-RFLP than that several bands generated using AFLP would produce
identical fingerprints.
13. Describe how you would identify the source organism responsible for generating a
particular band in a DGGE gel.
The identity of the source organism responsible for generating a particular band in a
DGGE gel can be determined by cutting out the band or a part of the band and purifying
the DNA. This DNA would then used as template in a second round of PCR using the
same primers (minus the GC clamp) that were used in the original amplification. The
resulting amplicon can be cloned or sequenced directly and the resultant sequence
compared to those in a sequence database (e.g., GenBank) using software such as
BLAST.
14. Describe what is meant by selection versus screening of a clone library. Explain
how the lacZ reporter works and incorporate its use in your description.
Selection is the method that allows one to determine what cells have been transformed
with the cloning vector, or plasmid carrying a selection marker, such as an antibiotic
resistance gene. Common antibiotic resistance markers used for cloning systems that use
the lacZ reporter are ampicillin and kanamycin. Thus, when the host cell is plated on a
medium that contains one of these antibiotics only those cells that contain the vector will
be able to grow.
Screening refers to the process by which vector containing cells are further analyzed to
determine which cells contain vectors in which the cloned DNA has successfully been
inserted. In the case of the lacZ reporter, the screening process is colorimetric where
white colonies are those that contain vectors with the insert and blue colonies are those
cells that contain the vector without an insert. The color difference is due in this case to
a process known as insertional inactivation. The cloning vector carries the first half of
the lacZ gene and the host cell carries the remainder. Internal to the vector-encoded lacZ
gene is the multiple cloning site. If a fragment of DNA is inserted into this site, this
portion of the lacZ gene no longer codes for a functional enzyme and the substrate in the
medium (X-Gal) cannot be cleaved so the colony remains white. When the functional
enzyme is formed the X-Gal is cleaved and a blue colonies result.