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Rapid Methods – Nucleic Acid Hybridization
1. Why use nucleic acid hybridization for detection of bacteria in foods?
Conventional methods for the selective growth & identification of significant microorganisms is based on culturing and isolation of the target microorganisms, possibly taking days to complete. The trend towards rapid methods is market driven: several days is too long to warehouse perishable commercial products for pathogen analysis; the cost in development of Nucleic acid and Immuno probes can represent a significant savings in storage and liability costs. There is a zero-tolerance standard for some pathogens (Salmonella & Listeria; even one-cell is not allowed per 25 gm sample). Also, it is signficantly more costly to recall product once it enters retail distribution compared to the ability to determine if food products contain pathogenic bacteria while still at hand. In addition, publicity attracted by an FDA- or USDA-mandated recall is costly to a company by potential loss of consumers.
The advancement of diagnostic abilities of nucleic acid technologies has shown promise in reducing the time required to make such determinations in foods. The time savings of many rapid methods is made by cutting into the time required for enrichment in conventional methods. Thus, rapidity means that detection is made with fewer numbers of cells- this is possible using nucleic acid or immuno probe technology.
Nucleic acid & immuno probes are not only beneficial to the commercial aspects of food industry, they also are very useful as diagnostic tools to aid in the understanding of dissemination of important DNA (virulence) sequences, mutations/changes in DNA sequences (are strains avirulent because they don't have genes for virulence, or do they have these sequences, but a mutation or otherwise is preventing expression of that characteristic?).
2. How is Nucleic Acid Hybridization Used?
In the most basic sense, nucleic acid hybridization utilizes the specific base pairing of nucleic acids (i.e., DNA) for their complementary sequence (i.e., DNA:DNA, RNA:DNA). The probe is labelled in some fashion and hybridized by any of several different methods to potential target DNA. Under stringent conditions the probe should ideally hybridize only to its target nucleic acid sequence. Depending of the method of labelling and the method of hybridization, detection of the probe (and therefore the target) via its label is attempted (Fig.1).
Types of hybridization/detection methods:
a) Hybridization in solution - Hybridization occurs in solution; detection can occur in solution too,
or after retaining the hybridized molecules onto filters.
b) Hybridization on filters - Hybridization can occur with target molecules initially bound onto nylon or nitrocellulose filters.
c) Hybridization in situ - Hybridization can occur in situ in tissues, or directly in agarose gels.
3. Target selection for nucleic acid hybridization
The choice of hybridization target (i.e., specific DNA, RNA, etc) depends on what you are wishing to detect. For instance, is the purpose to detect all members of a particular genus (i.e., all Salmonella), a particular species within a genus (i.e., Listeria monocytogenes but not other Listeria), or select strains within a given species (i.e., enterotoxigenic E. coli as opposed to non-toxigenic E. coli)? Therefore your choice of hybridization target will depend on the purpose intended of the hybridization assay.
What would you target in each of the three different purposes listed above?
Another factor to be considered is the copy per genome equivalent of the target molecule. A
chromosomal gene is normally expected to be found at one copy per cell. However, genes on
plasmids may be present in more than one copy depending on the copy-number of a particular
plasmid; similarly, ribosomal RNA targets are present in multiple copies within each cell.
In order to further increase the number of target molecules, one could include a strategy using a short period of either selective or nonselective enrichment to increase the number of (target)
microorganisms in the sample to be analyzed; this would indirectly increase the number of target molecules in the sample to be analyzed. Along similar lines, one could artificially increase the number of target molecules using a nucleic acid amplification scheme.
4. Selection and Labelling of ProbesA. Probes may consist of different types of molecules (need to be labelled before use):
1) Oligonucleotide probes - synthesized strands of nucleic acids which may range from 18-20
bases (i.e., 18-base oligomer, 18-mer) to 100 bases (100-mer). Single-stranded oligo probes,
obviously will only recognize (i.e., anneal) to one-half of the double-stranded target DNA. Also,
the shorter the probe, the greater the likelihood of non-specific hybridization reactions, that is,
binding to un-intended sites which randomly appear in the sequence of a target microorganisms'
DNA.
Labelled nucleotides can be incorporated during synthesis of the oligo probe. Labelling after
synthesis can be done at 5'-end (radiolabelling with phosphate) or at the 3'-end (with
radiolabelled/modified nucleotide triphosphates).
2) Cloned probes or gene fragments (double-stranded DNA) - plasmid clones, or isolated ds-
DNA fragments, may be used as probes. The non-essential portion of a plasmid vector
containing the probe may be used as long as the vector does not produce cross-reactions with
the target organisms, otherwise it would be necessary to isolate the particular DNA fragment
contained in the plasmid that may serve as a "probe."
Labelling of intact plasmid DNA can be done by nick translation, primer extension, or
photolabelling (biotin, digoxigenin). The efficiency of photolabelling is approximately one labe per 100-200 bases which would make it inefficent for short oligo probes. In nick translation, the
original DNA is labelled; in primer extension, new strands of labelled DNA are synthesized from
the original.
Large, linear ds-DNA fragments may also be labelled by nick translation, primer extension,
photolabelling, and 5'- and 3'-end labelling.
3) RNA probes - RNA can be transcribed from DNA in vitro, thereby generating large numbers of probe molecules. Labelling can be done during synthesis by supplying the appropriate labelled
nucleotide triphosphates or afterwards using photolabelling, etc.
B. Labels for Nucleic Acid Probes:1. Radiolabels - (32P, 35S, 3H, etc.)Radiolabels are usually incorporated into nucleic acids in the form of radionucleotides. The
radionucleotide triphosphate (dNTP) is either used to add a phosphate (-32P at 5' end) group or
a nucleotide monophosphate (-32P-dNMP at 3' end).
Advantages of radiolabels - high sensitivity of detection(can detect 0.1 pg DNA).
Disadvantage - short half-life of the nucleotide and the obvious problem of using radioactive
agents (handling, storage, disposal; prohibits use by nontechnical workers).
2. Modified Nucleotide TriphosphatesModified nucleotide triphosphates have been developed which have a modifying group attached
to the base. Having the labelling group attached to the base means that the method of labelling
must be one which adds a nucleotide, e.g., a synthesizing RXN (Nick Translation, Primer Extension).
i) Affinity - Biotinylated dNTP's (Biotin-dUTP). Avidin is a protein isolated from either egg white (egg white avidin) or from culture filtrates of Streptomyces avidinii (streptavidin). Avidin exhibits high affinity binding for biotin (Kd=10-15M). Avidin has been conjugated with either alkaline phosphatase (AP) or horseradish peroxidase (HRP). The avidin-enzyme conjugates are used to bind to the hybridized biotinylated DNA probe. Reaction of AP with the added dyes, NBT and BCIP, causes the production of an insoluble blue precipitate on a filter membrane. Alkaline phosphatase also reacts with chemiluminescent substrates which produces a
chemiluminescent reaction, emitting light, which can be detected with X-ray film.
ii) Immuno - Alternately, dNTP's can be conjugated with groups (digoxigenin; dNTP-dig) to which antibodies have been made. Therefore, anti-dig conjugated enzymes (Abdig-AP; Abdig-HRP) may be used to initiate a visualizing reaction wherever the dig-labelled probe is hybridized. Alkaline phosphatase may be used to produce either a chemiluminescent or colorimetric RXN.
3. Photoactivated labels
Photoactivated biotin and photoactivated digoxigenin can be bound to a DNA probe simply by
adding the DNA probe with the photoactivated biotin or dig and shining a light to cause a
crosslinking reaction. The probe is used to hybridize to its target, and the same conjugated
enzymes mentioned above (avidin-AP or Antidig-AP) can be used for visualization. Potency of
labelled probes - depends on the degree of labelling, since target detection depends on the
number of labels bound to the target sequence. For instance, end-labelled probes may contain
only one label per probe molecule. Since nick translation will replace a short stretch of 10-15
bases, a labelled nucleotide may be introduced several times within the stretch being repaired.
However, primer extension may synthesize a stretch of DNA as long as 600 bases within which
there is opportunity to introduce many labels within one strand of synthesized probe.
5. Hybridization of Nucleic Acids
A. Structure & biochemistry (see lecture 4)
The 2 polynucleotide strands of DNA are antiparallel; by convention, the DNA sequences are written
5' -> 3' with the 5'-P terminus at the left: P-5'-ATCGCG-3'-OH.
The helical structure of DNA (native state) is maintained by base stacking between adjacent bases and by hydrogen bonding between bases in opposite strands.
B. Factors affecting DNA denaturation
The 2 strands of DNA can be separated (denatured) by heat, high pH (pH 11.3), and low ionic
strength. Nucleic acids absorb light strongly at 260 nm; single stranded DNA has more absorbance than an equivalent amount of double stranded DNA because of a change in the electron systems of the bases in the stacked vs. unstacked form.
Tm= melting temperature at which denaturation is 50%.
Tm is dependent on ionic strength, base composition, and the presence of denaturing agents.
For instance:
i) Tm increases with increase in %GC composition due to 3 vs. 2 H-bonds for G:C.
ii) Tm increases as the ionic strength increases (0.15 - 0.5 M NaCl eliminates electrostatic repulsion between phosphate backbone of DNA strands).
iii) Tm is lowered by helix destabilizing reagents such as formamide which compete with bases for formation of H-bonds.
C. Factors Affecting the Rate of Denaturation
Concentration of probe - rate of hybridization to the filter and the amount of hybrids formed
increases with increasing concentration of the probe in solution.
Ionic strength (salt concentration) - at low ionic strength, hybridization occurs slowly, but as ionic strength increases (up to 1.5 M) so does the reaction rate. However, high salt concentrations stabilize mismatched duplexes.
Temperature - must be high enough to disrupt random intra- and inter-strand H-bonds that form, but low enough to allow renaturation (20-25oC below Tm).
D. Stringency of Hybridization Conditions
The discrimination of identical vs. mismatched sequences can be minimized by increasing
the stringency of hybridization. Hybridization conditions can be set which allow for
formation of hybrids with a high degree of mismatching (low stringency) or allow only
well-matched hybrids to form (high stringency). Low salt concentrations, high formamide concentrations, and high temperature favor formation of well-matched hybrids (high stringency).
6. Hybridization Strategies
Hybridization reactions can occur in either solid phase (on solid supports, i.e., filters) or in liquid
phase (i.e., in liquid). Solid phase, or filter, hybridization for detection of foodborne microorganisms is usually in the form of dot- or slot-blots, or with colony blots. In dot/slot blots,
bacterial cultures are lysed, releasing their DNA/RNA, and this solution is filtered through a
template of wells (i.e., dots) or slots. The nucleic acids are then trapped onto the nylon or
nitrocellulose filter which is then used in hybridization reactions; this gives a yes or no answer to the particular sample. Colony lifts are done by either placing filters down onto agar plates before plating a sample, or first plating a sample and picking up the colonies (i.e., colony lift) by placing a round filter onto the agar surface and picking it back up - the colonies will stick to the filter where they are lysed in situ and the filter is then used in hybridization reactions. These methods, however, are subject to high background signals that interfere with the probe signal.
Improved solid phase methods include:
Sandwich hybridization assay (Fig. 3).
Sandwich hybridization assay which involves a 3-component assay including a capture probe,
the target sequence (from the sample to be assayed), and a signal-generating probe (Fig. 3).
The denatured target sequence (sample) is retained during hybridization by the capture
probe (bound to the solid phase support) and the signal probe is then added and hybridizes to an
alternate sequence on the target that is not involved with binding to the capture probe. This
can be considered a double capture: once for the target, and a second time for the signal probe.
Strand-displacement assay (Fig. 4).
A next generation modification of the sandwich assay is
the strand-displacement assay (Fig. 4). This assay also involves a capture probe to which is
hybridized a signal probe that has been made to have some degree of mismatching and is
therefore weakly hybridized to the capture probe.
When a target sequence of 100% identity to the capture probe is hybridized, it displaces the wealy bound signal probe. Detection of target sequence is obtained by a decrease or absence of signal.
Dipstick assay (Fig. 5).Another variation of the solid-phase assay is the dipstick assay (Fig. 5). This assay is nearly
identical to the sandwich assay described earlier (Fig. 3). However, in the dipstick assay hybridization occurs in solution and the capture probe is not directly attached to
a solid support. Rather, one end of the capture probe is for "capturing" the target sequence while the other end is for attachment by hybridization to complementary sequences attached to a dipstick. The signal also binds to the target sequence (captured), however, detection is
made by means of an antibody-enzyme conjugate that recognizes the signal label. Other hybridization strategies have also made use of magnetic bead technology. The capture probe can be attached to magnetic beads which are allowed to hybridize with potential target molecules.
The capture:target hybrids are removed from solution by magnetic recovery; addition of a signal probe and subsequent washing and signal development provides a detection signal.
7. Amplification Methods
Even with all the sensitiviy of DNA probes, one of the major obstacles is when there occurs only a few target cells against a high background of other organisms and food debri. One method of improving the chances for target detection is by amplification.
Polymerase Chain Reaction (PCR)
PCR is a method which has gained tremendous popularity for its ability to detect low levels of DNA sequences. Many of the advances in biotechnology/molecular biology come from a simple understanding of the mechanics of the various reactions involved with DNA synthesis/ degradation. PCR is simply a repetition of primer extension reactions (Fig. 6). Two synthetic primers made to complementary sequences of target DNA are annealed to denatured target DNA. Taq polymerase isolated from Thermus aquaticus, an organism capable of tolerating high thermal temperatures, is used instead of DNA polymerase. What this does is allow high temperatures to be reached without denaturing the polymerase enzyme. Primer extension (strand synthesis) is carried out at moderately high temperatures as well (~55oC), the two facing primers are extended, the temperature is raised to ~95oC to dislodge the extended strands, the temperature is then lowered and new primers (in excess) reanneal and repeat the strand extension process.
The thermal stability of Taq polymerase provides 3 advantages:
a) Faster RXN rate of synthesis
b) Prevents ssDNA from selfannealling
c) Allows the repetitive denaturing of DNA without denaturing the polymerase.
The strands that are synthesized and extended can also act as template for the primers, thereby exponentially increasing the synthesis of the target sequences and thereby facilitating the amplification. The novelty/utility in PCR is the ability to amplify sequences by a factor of up to 105-fold.
Primer Development Strategy:
Primer Tm. One aspect of primer development is to insure the two primers have similar Tm. At
any given temperature, you would want both primers to have similar potential for hybridization which would not occur if they had vastly different Tm’s. Software programs can now select primers for nearly the same Tm. Primer Self-hybridization. Also, primers should not have excessive cross hybridization with themselves or the other primer. This would prevent the primers from hybridizing efficiently to their target. If primers were to self-overlap, you could also get primer-dimers to develop upon PCR reactions. Software programs can also determine if this will likely occur.
Q-beta Replicase Amplification:
This method is again a double-capture hybridization assay (Fig. 7). Denatured target sequence is captured by immobilized capture probe. An RNA probe (MDV-1) is then added which can also
recognize the target sequence. The immobilized mixture is then washed and unbound RNA probe is washed away except for that retained by the captured target sequence. The target-RNA probe is then selectively eluted and an RNA-dependent RNA polymerase (Q-beta replicase) is added. The Q-beta polymerase synthesizes RNA transcripts from RNA
(the one used as the probe & stuck to the target). Amplification occurs very rapidly to give amplification levels of up to 109-fold.
Ligase Amplification Reaction (LAR):
LAR is similar, yet different, than PCR. Instead of synthesizing new strands of DNA as in PCR, LAR relies on the use of adjacent annealing oligonuclceotides (Fig. 8). When they hybridize next
to each other on target sequence, they can be ligated together by the enzyme, T4 DNA ligase. The same thing happens on the opposite strand (therefore, you actually need 4 oligos). The
newly ligated oligos can then act as template for the hybridization of the two complementary
oligos, and so on, etc. This technique can give up to 106-fold amplification of target sequence.
Self-Sustained Sequence Replication (3SR)
3SR is an attempt to get away from thermal cycling because Cetus had patented the PCR process (now sold the patent to Hoffman- LaRoche) and anyone who uses the process,
commercially, has to pay royalties. 3SR makes use of a combination of reverse transcriptase
(makes DNA from RNA), RNA polymerase (makes RNA from DNA), and RNase H (degrades RNA when hybridized to DNA) (Fig. 9). In this process, RNA targets are degraded
and result in an accumulation (amplification) of DNA. Reverse transcriptase is a DNA
polymerase that can synthesize a complementary copy of DNA from a single strand of RNA, DNA, or an RNA:DNA hybrid.
The result of using Reverse Transcriptase in the 3SR assay would be double stranded DNA from
the original single stranded RNA target.
Steps:
1. Primers for the RNA target are added that initiate synthesis of DNA from the RNA by reverse transcriptase.
2. RNase H (Rev. Transcript.) is added which degrades the RNA in an RNA:DNA hybrid.
3. A second primer is added which allows synthesis of a complementary strand of DNA (dsDNA) from the remaining DNA of the DNA:RNA hybrid.
4. The double-stranded DNA can then be assayed, or used to further amplification by:
5. Addition of primers with RNA polym. binding site, and the use of RNA polymerase to
transcribe an RNA copy (both directions). RNA doesn't need a priming site as in
DNAsynthesis, but it does need an RNA polymerase binding site.
6. After transcriptional replication of the RNA strands, DNA amplification from these strands
will continue just as in Step 1.
Fluorogenic PCR Assays for Rapid and Real-Time PCR.
The Polymerase Chain Reaction (PCR) permits the rapid amplification of specific DNA sequences by a factor of up to 107 (Border et al., 1990). The sensitivity of PCR is typically compared to the detection of a single bacterium. Although carrying out the PCR involves initial optimization, these efforts are more than justified by the sensitivity and the wide dynamic range of the technique. During the past 10 years PCR has been used to detect foodborne pathogens from various food products such as milk, cheese, eggs, meat and meat products, fish and fish
6 products. Analysis of the PCR product has usually been done using ethidium bromide-stained agarose gels. However, the use of fluorogenic probes that generate a fluorescent signal can eliminate the need to run agarose gels for PCR analyses such that automation of pathogen detection may be applied more easily in the food processing industry.
Hybridization probes.
The Light Cycler™ can also use hybridization probes for more specific detection of the product (De Silva et al. 1998). The Light Cycler™ uses the concept of Fluorescence Resonance Energy Transfer (FRET) wherein fluorescence energy transfer occurs between two adjacent
fluorophores (De Silva et al. 1998) (Fig.12). The probes are designed such that they are specific to an internal sequence within the target. The 3’ end of one probe is labeled with a donor fluorophore (fluorescein) and the 5’ end of the adjacent probe is labeled with an acceptor fluorophore (LC Red 640) (Bernard et al. 1999). In order to prevent the extension of the probe
labelled with LC Red 640, its 3’ end is blocked by dephopsphorylation. The Tm of the probes should be 5.0 0C to 10.0 0C higher than the Tm of the primers (Bernard et al. 1999). The donor fluorophore absorbs light from the blue light-emitting diode (LED) of the Light Cycler™ instrument, the resonance energy from this fluorophore is absorbed by the adjacent acceptor fluorophore and the fluorescence emitted by it is detected and measured (De Silva et al. 1998). This energy transfer occurs only when the two probes are in close proximity (i.e bound to the target). The FRET signal increases with each thermal cycle and is proportional to the amount of specific PCR product available for hybridization (De Silva et al. 1998). Though the sensitivity of the technique is high the method requires designing, synthesis and optimization of the hybridization probes in addition to the optimization of the PCR.
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