POLYMERASE CHAIN POLYMERASE CHAIN REACTIONREACTION

PCRPCR

Amplification of a specific DNA sequence Amplification of a specific DNA sequence (100-5000 bp)(100-5000 bp)

2 synthetic oligonucleotide primers 2 synthetic oligonucleotide primers flanking the target sequenceflanking the target sequence

Use of thermostable DNA polymeraseUse of thermostable DNA polymerase 3 step cycling process :3 step cycling process :

-denaturation-denaturation

-annealing of primers-annealing of primers

-extension-extension

Repeated cycle

historyhistory

Kary MullisKary Mullis 1st thermal cycler :19861st thermal cycler :1986 3 waterbaths with different 3 waterbaths with different

temperature, non thermostable DNA temperature, non thermostable DNA polymerasepolymerase

Polymerase from Polymerase from Thermus aquaticusThermus aquaticus (Taq polymerase) : Topt:72C,withstand (Taq polymerase) : Topt:72C,withstand heating to 100Cheating to 100C

Other polymerase: PfuOther polymerase: Pfu

DENATURATIONDENATURATION

Denaturation: heating DNA to temperature above the Tm to Denaturation: heating DNA to temperature above the Tm to make ssDNA. make ssDNA.

if the DNA is heated in buffers of ionic strength lower if the DNA is heated in buffers of ionic strength lower than than 150mM NaCl150mM NaCl, the melting temperature is generally less , the melting temperature is generally less than 100than 100ooC - which is why PCR works with C - which is why PCR works with denaturing denaturing temperatures of 91-97temperatures of 91-97ooCC. .

Taq polymeraseTaq polymerase  has ahas a  half-life of 30 min at 95half-life of 30 min at 95ooCC, one , one should not do more than about should not do more than about 30 amplification cycles30 amplification cycles: : however, it is possible to however, it is possible to reduce the denatureduce the denaturration ation temperaturetemperature after about 10 rounds of amplification, as  after about 10 rounds of amplification, as the the mean length of target DNA is decreasedmean length of target DNA is decreased: for templates : for templates of of 300bp or less300bp or less, denaturation temperature may be reduced , denaturation temperature may be reduced to to as low as 88as low as 88ooC for 50% (G+C)C for 50% (G+C) templates (Yap and McGee,  templates (Yap and McGee, 1991), which means one may do as many as 1991), which means one may do as many as 40 cycles40 cycles without  without much decrease in enzyme efficiency. much decrease in enzyme efficiency.

DENATURATIONDENATURATION

""Time at temperatureTime at temperature" is the main reason " is the main reason for denaturation / loss of activity of Taq: thus, for denaturation / loss of activity of Taq: thus, if one reduces this, one will if one reduces this, one will increase the increase the number of cycles that are possiblenumber of cycles that are possible, , whether the temperature is reduced or not. whether the temperature is reduced or not.

Normally the denaturation time is Normally the denaturation time is 1 min at 1 min at 9494ooCC: it is possible, for short template : it is possible, for short template sequences, to sequences, to reduce this to 30 sec or reduce this to 30 sec or lessless. .

Increase in denaturation temperature and Increase in denaturation temperature and decrease in time may also work: Innis and decrease in time may also work: Innis and Gelfand (1990) recommend Gelfand (1990) recommend 9696ooC for 15 secC for 15 sec. .

annealingannealing

T T meltingmelting : depend on : depend on Primer length and Primer length and sequencesequence, , the melting temperature of a DNA the melting temperature of a DNA duplex increases with its length and (G+C) content duplex increases with its length and (G+C) content

a simple formula for calculation of thea simple formula for calculation of the primers primers Tm is Tm is Tm = 4(G + C) + 2(A + Tm = 4(G + C) + 2(A + T)T)ooCC

annealing temperature (Ta) about annealing temperature (Ta) about 55ooC below the C below the lowest Tm of the pair of primers to be lowest Tm of the pair of primers to be used used (Innis and Gelfand, 1990)(Innis and Gelfand, 1990)

if the Ta is increased by 1if the Ta is increased by 1ooC every other cycle, C every other cycle, specificity of amplification and yield of specificity of amplification and yield of products <1kb in length are both increasedproducts <1kb in length are both increased

annealingannealing

If a Ta is too low : one or both primers willIf a Ta is too low : one or both primers will anneal to sequences other than the anneal to sequences other than the true target, true target, as internal single-base as internal single-base mismatches or partial annealing may be mismatches or partial annealing may be tolerated tolerated 

this is fine if one wishes to amplify this is fine if one wishes to amplify similar or related targetssimilar or related targets; however, it ; however, it can lead to can lead to "non-specific" amplification "non-specific" amplification and consequent reduction in yield and consequent reduction in yield of of the desired productthe desired product

annealingannealing

too high Ta : too high Ta : too little product will be too little product will be mademade, as the likelihood of primer annealing is , as the likelihood of primer annealing is reduced reduced

  a pair of primers with very different Tas a pair of primers with very different Tas may may never give appreciable yields of a unique never give appreciable yields of a unique product,product, and may also result in inadvertent  and may also result in inadvertent ""asymmetricasymmetric" or single-strand amplification " or single-strand amplification of the most efficiently primed product strand.of the most efficiently primed product strand.

Annealing does not take long:Annealing does not take long: most primers  most primers will will anneal efficiently in 30 sec or lessanneal efficiently in 30 sec or less, unless the , unless the Ta is too close to the Tm, or unless they are Ta is too close to the Tm, or unless they are unusually long unusually long    

An illustration of the effect of annealing temperature on the specificity and on the yield of amplification of Human papillomavirus type 16 (HPV-16) (Williamson and Rybicki, 1991: J Med Virol 33: 165-171)

Plasmid and biopsy sample DNA templates were amplified at different annealing temperatures as shown: note that while plasmid is amplified from 37 to 55oC, HPV DNA is only specifically amplified at 50oC.

EEXTENSIONXTENSION

normally 70 - 72oC, for 0.5 - 3 minnormally 70 - 72oC, for 0.5 - 3 min elongation occurs from the moment of elongation occurs from the moment of

annealing,annealing,  aat around 70oC the activity is t around 70oC the activity is optimal, andoptimal, and primer extension occurs at up to primer extension occurs at up to 100 bases/sec100 bases/sec

About About 1 min is sufficient for reliable 1 min is sufficient for reliable amplification of 2kb sequences amplification of 2kb sequences (Innis and (Innis and Gelfand, 1990). Longer products require longer Gelfand, 1990). Longer products require longer times: times: 3 min is a good bet for 3kb and longer 3 min is a good bet for 3kb and longer productsproducts. Longer times may also be helpful in . Longer times may also be helpful in later cycles when product concentration exceeds later cycles when product concentration exceeds enzyme concentration (>1nM), and when dNTP enzyme concentration (>1nM), and when dNTP and / or primer depletion may become limiting. and / or primer depletion may become limiting.

REACTION BUFFERREACTION BUFFER

Recommended buffers generally contain :Recommended buffers generally contain : 10-50mM Tris-HCl pH 8.3,10-50mM Tris-HCl pH 8.3, up to 50mM KCl, 1.5mM or higher MgCl2,up to 50mM KCl, 1.5mM or higher MgCl2, primers 0.2 - 1uM each primer,primers 0.2 - 1uM each primer, 50 - 200uM each dNTP,50 - 200uM each dNTP, gelatin or BSA to 100ug/ml,gelatin or BSA to 100ug/ml, and/or non-ionic detergentsand/or non-ionic detergents such  such

as as Tween-20 or Nonidet P-40 or Triton X-Tween-20 or Nonidet P-40 or Triton X-100100 (0.05 - 0.10% v/v) (0.05 - 0.10% v/v)

(Innis and Gelfand, 1990).  Modern formulations (Innis and Gelfand, 1990).  Modern formulations may differ considerablymay differ considerably

REACTION BUFFERREACTION BUFFER

Higher than 50mM KCl or NaCl inhibits Higher than 50mM KCl or NaCl inhibits Taq,Taq, but some is necessary to facilitate primer  but some is necessary to facilitate primer annealingannealing

[Mg2+] affects primer annealing; [Mg2+] affects primer annealing; Tm of Tm of template, product and primer-template template, product and primer-template associations; product specificity; enzyme activity associations; product specificity; enzyme activity and fidelity. Taq requires and fidelity. Taq requires freefree Mg2+, so allowances  Mg2+, so allowances should be made for dNTPs, primers and template, should be made for dNTPs, primers and template, all of which chelate and sequester the cation; of all of which chelate and sequester the cation; of these, dNTPs are the most concentrated, so [Mg2+] these, dNTPs are the most concentrated, so [Mg2+] should be 0.5 - 2.5mM should be 0.5 - 2.5mM greatergreater than [dNTP].  than [dNTP]. A A titration should be performed with varying titration should be performed with varying [Mg2+] with all new template-primer [Mg2+] with all new template-primer combinationscombinations, as these can differ markedly in , as these can differ markedly in their requirements, even under the same conditions their requirements, even under the same conditions of concentrations and cycling times/temperatures. of concentrations and cycling times/temperatures.

REACTION BUFFERREACTION BUFFER

Some enzymes do not need added Some enzymes do not need added protein,protein, others are dependent on it. Some  others are dependent on it. Some enzymes work markedly better in the enzymes work markedly better in the presence of detergent, probably because it presence of detergent, probably because it prevents the natural tendency of the prevents the natural tendency of the enzyme to aggregate. enzyme to aggregate.

Primer concentrations should not go Primer concentrations should not go above 1uM above 1uM unless there is a high degree unless there is a high degree of degeneracy; 0.2uM is sufficient for of degeneracy; 0.2uM is sufficient for homologous primers. homologous primers.

Nucleotide concentrationNucleotide concentration need not be  need not be above 50uM each: long products may above 50uM each: long products may require more, however. require more, however.

Cycle NumberCycle Number The number of amplification cycles necessary to The number of amplification cycles necessary to

produce a band visible on a gel depends largely on produce a band visible on a gel depends largely on the starting concentration of the target DNAthe starting concentration of the target DNA  

Innis and Gelfand (1990) recommend from Innis and Gelfand (1990) recommend from 40 - 45 cycles 40 - 45 cycles to amplify 50 target moleculesto amplify 50 target molecules, and , and 25 - 30 to amplify 25 - 30 to amplify 3x105 molecules3x105 molecules to the same concentration.  to the same concentration.

This non-proportionality is due to a so-called This non-proportionality is due to a so-called plateau plateau effecteffect, which is the attenuation in the exponential rate of , which is the attenuation in the exponential rate of product accumulation in late stages of a PCR, when product product accumulation in late stages of a PCR, when product reaches 0.3 - 1.0 nM. This may be caused by degradation of reaches 0.3 - 1.0 nM. This may be caused by degradation of reactants (dNTPs, enzyme); reactant depletion (primers, reactants (dNTPs, enzyme); reactant depletion (primers, dNTPs - former a problem with short products, latter for dNTPs - former a problem with short products, latter for long products); end-product inhibition (pyrophosphate long products); end-product inhibition (pyrophosphate formation); competition for reactants by non-specific formation); competition for reactants by non-specific products; competition for primer binding by re-annealing of products; competition for primer binding by re-annealing of concentrated (10nM) product (Innis and Gelfand, 1990). concentrated (10nM) product (Innis and Gelfand, 1990).

If desired product is not made in 30 cycles, take a small sample (1ul) of the amplified mix and re-amplify 20-30x in a new reaction mix rather than extending the run to more cycles: in some cases where template concentration is limiting, this can give good product where extension of cycling to 40x or more does not

nested primer PCRnested primer PCR

PCR amplification is performed with one PCR amplification is performed with one set of primers, then some product is set of primers, then some product is taken - with or without removal of taken - with or without removal of reagents - for re-amplification with an reagents - for re-amplification with an internally-situated, "nested" set of internally-situated, "nested" set of primers.  This process adds another primers.  This process adds another level of specificity, meaning that all level of specificity, meaning that all products non-specifically amplified in products non-specifically amplified in the first round will not be amplified in the first round will not be amplified in the second the second

This gel photo shows the effect of nested PCR amplification on the detectability of Chicken anaemia virus (CAV) DNA in a dilution series: the PCR1 just detects 1000 template molecules; PCR2 amplifies 1 template molecule (Soiné C, Watson SK, Rybicki EP, Lucio B, Nordgren RM, Parrish CR, Schat KA (1993)  Avian Dis 37: 467-476).

Helix Destabilisers / Helix Destabilisers / AdditivesAdditives

With NAs of high (G+C) content, it may beWith NAs of high (G+C) content, it may be necessary necessary to use harsher denaturation conditions. For to use harsher denaturation conditions. For example, one may incorporate up to example, one may incorporate up to 10% (w or 10% (w or v/v) :v/v) :

dimethyl sulphoxide (DMSO),dimethyl sulphoxide (DMSO), dimethyl formamide (DMF),dimethyl formamide (DMF), ureaurea or formamide or formamide 

These additives are presumed to lower the Tm of the target NA, although DMSO at 10% and higher is known to decrease the activity of Taq by up to 50% (Innis and Gelfand, 1990; Gelfand and White, 1990).

Additives may also be necessary in the amplification of long target sequences: DMSO often helps in amplifying products of >1kb. Formamide can apparently dramatically improve the specificity of PCR (Sarkar et al., 1990), while glycerol improves the amplification of high (G+C) templates (Smith et al., 1990)

Polyethylene glycol (PEG) may be a useful additive when DNA template concentration is very low: it promotes macromolecular association by solvent exclusion, meaning the pol can find the DNA

A simple set of rules for primer sequence A simple set of rules for primer sequence design (adapted from Innis and Gelfand, 1991):design (adapted from Innis and Gelfand, 1991):

primers should be 17-28 bases in length;primers should be 17-28 bases in length; base composition should be 50-60% (G+C);base composition should be 50-60% (G+C); primers should end (3') in a G or C, or CG or GC: this primers should end (3') in a G or C, or CG or GC: this

prevents "breathing" of ends and increases efficiency of prevents "breathing" of ends and increases efficiency of priming;priming;

Tms between 55-80Tms between 55-80ooC are preferred;C are preferred; runs of three or more Cs or Gs at the 3'-ends of primers runs of three or more Cs or Gs at the 3'-ends of primers

may promote mispriming at G or C-rich sequences may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be (because of stability of annealing), and should be avoided;avoided;

3'-ends of primers should not be complementary (ie. base 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised pair), as otherwise primer dimers will be synthesised preferentially to any other product;preferentially to any other product;

primer self-complementarity (ability to form primer self-complementarity (ability to form SECONDARY SECONDARY structures such as hairpins) should be avoided.structures such as hairpins) should be avoided.

PRIMER DESIGNPRIMER DESIGN

Requires knowledge of some Requires knowledge of some sequencesequence informationinformation

Gives high specificity and sensitivityGives high specificity and sensitivity Allows amplification from limited Allows amplification from limited

startingstarting materialmaterial

APPLICATION OF PCRAPPLICATION OF PCR

DIAGNOSTICDIAGNOSTIC BIODIVERSITY ANALYSISBIODIVERSITY ANALYSIS LABORATORY ROUTINELABORATORY ROUTINE

Diagnostic Applications of Diagnostic Applications of PCRPCR

detecting pathogens using genome-detecting pathogens using genome-specific primer pairsspecific primer pairs

screening specific genes for unknown screening specific genes for unknown mutationsmutations

genotyping using known STS genotyping using known STS (Sequence Tagged Sites)(Sequence Tagged Sites) markersmarkers

Laboratory applications of Laboratory applications of PCRPCR

subcloning DNA targets using PCR,subcloning DNA targets using PCR,

--T/A CloningT/A Cloning

--Restriction Site AdditionRestriction Site Addition

--Blunt-end Ligation Blunt-end Ligation PCR-mediated in vitroPCR-mediated in vitro mutagenesis.mutagenesis.

Reverse transcription Reverse transcription polymerase chain reaction polymerase chain reaction

Amplification from mRNA templateAmplification from mRNA template RNA strand is first reverse transcribed into its DNA complement RNA strand is first reverse transcribed into its DNA complement

((complementary DNAcomplementary DNA, or , or cDNAcDNA) using the enzyme reverse ) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional transcriptase, and the resulting cDNA is amplified using traditional PCR PCR

The two-step RT-PCR process for converting RNA to DNA and the subsequent PCR amplification of the reversely-transcribed DNA:

First strand reaction: complementary DNA (cDNA) is made from an mRNA template using dNTPs & reverse transcriptase. The components are combined with a DNA primer in a reverse transcriptase buffer for an hour at 42°C.

Second strand reaction: after the reverse transcriptase reaction is complete, cDNA has been generated from the original ss mRNA, standard PCR (called the “second strand reaction”) is initiated.

In the two-step RT-PCR a thermostable DNA polymerase & the upstream and downstream DNA primers are added. Heating the reaction to temperatures above 37°C facilitates binding of DNA primers to the cDNA, & subsequent higher temperatures allow the DNA polymerase to make double-stranded DNA from the cDNA. Heating the reaction to ~95°C melts the two DNA strands apart, enabling the primers to bind again at lower temperatures and begin the chain reaction again. After ~30 cycles, millions of copies of the sequence of interest are generated

The use of RT-PCRThe use of RT-PCR the diagnosis of genetic diseases the diagnosis of genetic diseases tthe determination of the abundance of specific different he determination of the abundance of specific different

RNA molecules within a cell or tissue as a measure RNA molecules within a cell or tissue as a measure of gene expressionof gene expression

the cloning of eukaryotic genes in prokaryotes the cloning of eukaryotic genes in prokaryotes most eukaryotic genes contain introns which are present most eukaryotic genes contain introns which are present

in the genome but not in the mature mRNA, the cDNA in the genome but not in the mature mRNA, the cDNA generated from a RT-PCR reaction is the exact DNA generated from a RT-PCR reaction is the exact DNA sequence which would be directly translated into protein sequence which would be directly translated into protein after transcription. When these genes are expressed in after transcription. When these genes are expressed in prokaryotic cells such as E. coli for protein prokaryotic cells such as E. coli for protein production/purification, the RNA produced directly from production/purification, the RNA produced directly from transcription need not undergo splicing as the transcript transcription need not undergo splicing as the transcript contains only exons (prokaryotes lack the mRNA splicing contains only exons (prokaryotes lack the mRNA splicing mechanism of eukaryotes) mechanism of eukaryotes)

studying the genomes of viruses whose genomes are studying the genomes of viruses whose genomes are composed of RNA, such as retroviruses like HIV composed of RNA, such as retroviruses like HIV

Real-time polymerase chain reaction Real-time polymerase chain reaction

amplify and simultaneously quantify a amplify and simultaneously quantify a targeted DNA moleculetargeted DNA molecule. . measurement of DNA measurement of DNA amplification during PCR in real time, i.e., the amplification during PCR in real time, i.e., the amplified product is measured at each PCR cycle amplified product is measured at each PCR cycle

  Two common methods of quantification are: (1) Two common methods of quantification are: (1) the use of fluorescent dyes that intercalate with the use of fluorescent dyes that intercalate with double-stranded DNA, and (2) modified double-stranded DNA, and (2) modified DNAoligonucleotide probes that fluoresce when DNAoligonucleotide probes that fluoresce when hybridized with a complementary DNA hybridized with a complementary DNA

Frequently, real-time Frequently, real-time polymerase chain reaction polymerase chain reaction is combined with reverse transcription to is combined with reverse transcription to quantify messenger RNA (mRNA) in cells or quantify messenger RNA (mRNA) in cells or tissuestissues

Real-time PCR using double-Real-time PCR using double-stranded DNA dyes stranded DNA dyes

A DNA-binding dye binds to all dsDNA in PCR, causing A DNA-binding dye binds to all dsDNA in PCR, causing fluorescence of the dye. An increase in DNA product during fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA and is measured at each cycle, thus allowing DNA concentrations to be quantified. However, dsDNA dyes such concentrations to be quantified. However, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products, including as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as "primer dimers"). This can nonspecific PCR products (such as "primer dimers"). This can potentially interfere with or prevent accurate quantification potentially interfere with or prevent accurate quantification of the intended target sequenceof the intended target sequence

The reaction is run in a thermocycler, and after each cycle, The reaction is run in a thermocycler, and after each cycle, the levels of fluorescence are measured with a detector; the the levels of fluorescence are measured with a detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR dye only fluoresces when bound to the dsDNA (i.e., the PCR product). With reference to a standard dilution, the dsDNA product). With reference to a standard dilution, the dsDNA concentration in the PCR can be determined concentration in the PCR can be determined

SYBR Green can’t bind single stranded DNA or primer

But it can bind double stranded DNA once the polymerase has made the 2nd strand

Fluorescent reporter probe Fluorescent reporter probe method method

uses a sequence-specific RNA or DNA-based uses a sequence-specific RNA or DNA-based probe to quantify only the DNA containing probe to quantify only the DNA containing the probe sequence; therefore, use of the the probe sequence; therefore, use of the reporter probe significantly increases reporter probe significantly increases specificity specificity

allows quantification even in the presence of allows quantification even in the presence of some non-specific DNA amplification some non-specific DNA amplification

allows for multiplexing - assaying for several allows for multiplexing - assaying for several genes in the same reaction by using specific genes in the same reaction by using specific probes with different-coloured labels, probes with different-coloured labels, provided that all genes are amplified with provided that all genes are amplified with similar efficiency similar efficiency

commonly carried out with an RNA-based probe with commonly carried out with an RNA-based probe with a fluorescent reporter at one end and a fluorescent reporter at one end and a quencher of fluorescence at the opposite end of a quencher of fluorescence at the opposite end of the probe. The close proximity of the reporter to the probe. The close proximity of the reporter to the quencher prevents detection of its the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of the taq 3' exonuclease activity of the taq polymerase breaks the reporter-quencher polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of proximity and thus allows unquenched emission of fluorescence, which can be detected. An increase fluorescence, which can be detected. An increase in the product targeted by the reporter probe at in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of increase in fluorescence due to the breakdown of the probe and release of the reporter the probe and release of the reporter

The PCR is prepared as usual and the reporter probe is added.

As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target.

Polymerisation of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5'-3-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence.

Fluorescence is detected and measured in the real-time PCR thermocycler, and its geometric increase corresponding to exponential increase of the product is used to determine the threshold cycle (CT) in each reaction.

(1) In intact probes, reporter fluorescence is quenched.(2) Probes and the complementary DNA strand are

hybridized and reporter fluorescence is still quenched.(3) During PCR, the probe is degraded by the Taq

polymerase and the fluorescent reporter released.

Principle of quantitationPrinciple of quantitation The number of cycles it takes to reach a certain amount of The number of cycles it takes to reach a certain amount of

fluorescence is proportional to the amount of cDNA present at the fluorescence is proportional to the amount of cDNA present at the start start

This can be plotted as cycle no v log concentration to give a This can be plotted as cycle no v log concentration to give a straight linestraight line

By using known amounts of cDNA you can obtain a standard curve By using known amounts of cDNA you can obtain a standard curve

to find out levels of an unknownto find out levels of an unknown

The use of real time PCRThe use of real time PCR rapidly detect the presence of genes involved rapidly detect the presence of genes involved

in infectious diseases, cancer and genetic in infectious diseases, cancer and genetic abnormalitiesabnormalities

determining how the genetic expression of a determining how the genetic expression of a particular gene changes over time, such as in particular gene changes over time, such as in the response of tissue and cell cultures to an the response of tissue and cell cultures to an administration of a pharmacological agent, administration of a pharmacological agent, progression of cell differentiation, or in response progression of cell differentiation, or in response to changes in environmental conditionsto changes in environmental conditions

used in environmental microbiology, for used in environmental microbiology, for example to quantify resistance genes in water example to quantify resistance genes in water samples samples