application note a08-005a detecting low copy numbers

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Application Note A08-005A

Detecting low copy numbers

Introduction Sensitivity of a qPCR assay is highly dependent on primer efficiency. Not all assays will

be capable of detecting a single copy of template in a reaction due to non-specific

events which may limit the sensitivity of the assay. However all qPCR instruments

should theoretically be able to detect a single copy of target amplified at 100%

efficiency within 40 cycles. This is the point at which the copy is amplified to such an

extent that the fluorescent signal produced by the product exceeds the detection

threshold of the instrument and can be determined above the background of the no

template control samples.

Methods In order to determine as accurately as possible the copy number of the sample, a template of known size was

quantified using a sensitive fluorimetric assay. The template used was Lambda phage DNA (Promega, part code

D150A) which has a genome size of 48502 bp. The number of copies of template can be calculated using the

following formula:

DNA (copies) = 6.022 x 1023

(copies mol-1

) x DNA amount (g)

DNA length (bp) x 660 (g mol-1

bp-1

)

Therefore, each µg of Lambda DNA contains 1.88 x 1010

copies of the genome.

Fluorimetric assay

Lambda template DNA

Calf thymus DNA standard 1mg/ml in TE, pH 8.0

10,000 x SYBR® Green I solution

TE buffer, pH 8.0

The standard DNA was diluted in TE buffer to give a solution of 0.1ng/µl. The 10,000x SYBR® Green solution was

also diluted in TE buffer to give a 2x solution. Various volumes of standard or sample were pipetted into wells of a

96-well PCR plate together with the indicated amounts of TE buffer and SYBR® Green solution to give a range of

standards ranging from 0 to 10ng DNA. The plate was sealed and the fluorescence measured in PrimeQ at 50°C.

Each standard and sample was prepared in triplicate and the fluorescence read 10 times.

DNA (ng) Vol. 0.1ng/µl standard (µl) Vol. TE (µl) Vol. 2x SYBR® Green (µl)

10 100 0 100

8 80 20 100

6 60 40 100

4 40 60 100

2 20 80 100

0 0 100 100

UNK1 50 50 100

UNK2 40 40 100

Table 1: Preparation of standards and samples for quantification.

A standard curve was constructed from the fluorescence values of the standards and used to calculate the

concentration of the unknown Lambda DNA samples.

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A08-005A: Detecting low copy numbers

PCR

Lambda template DNA

10x primer mix

GoTaq® QPCR Master Mix (2x) (Promega, part code A6001)

Nuclease-free water

The primers were designed using Primer-BLAST (1) to amplify a 155bp target of the Lambda DNA. Details of the

primers are given in Table 2.

Primer name Sequence (5’-3’) Product length (bp) Final conc. in assay Tm used

Lambda 15 For TGCTGCCCTGCTGACGCTTC 155

0.3µM 65°C

Lambda 15 Rev GTGCAGACAGCTGGCGACGT 0.3µM

Table 2: Lambda primers.

The template concentration was adjusted to 0.2x107 copies/µl (5µl = 1x10

7 copies) based on the results of the

fluorimetric assay and using the formula above to calculate the copy number. This was then serially diluted in 1 in

10 in nuclease-free water until reaching 0.2 copies/µl (5µl = 1 copy). Four separate master mixes were prepared,

each sufficient for 12 replicates at each of 100 copies, 10 copies and 1 copy per well of the template DNA and 12

replicates of NTCs. 20µl reactions were used. The samples were amplified according to the thermal cycling program

shown in Table 3.

Stage Number of Cycles Step Temp Time Read/Filter

Enzyme Activation 1 95°C 2 min

Amplification 50

Denaturation 94°C 15 sec

Anneal 65°C 20 sec

Extend 72°C 20 sec FC02, FC04,

Ramp 31 (0.5°C intervals) Ramp 80-95°C 10 sec FC02

Table 3: Thermal cycling program for the Lambda qPCR assay.

Following real-time PCR collection of data, the samples were run on 2% agarose gels to confirm the presence or

absence of PCR products.

Results The concentration of the Lambda phage DNA was determined using a quantitative fluorescence assay using the

intercalating dye SYBR® Green I. Standards and samples were read in a PCR plate using PrimeQ as a plate reader to

obtain the fluorescence values. The standard curve is shown in Figure 1. Using this curve the concentration of

Lambda DNA was determined and the number of copies calculated using the formula described above.

y = 3868.5x + 11070R² = 0.9982

0

10000

20000

30000

40000

50000

60000

0 2 4 6 8 10

RF

U

ng DNA

DNA standard curve

Figure 1: Fluorescence assay DNA standard curve.

The fluorescence was measured by addition of

SYBR® Green I solution to the standard DNA and

detected in PrimeQ. Values are the average of

triplicates, each read 10 times.



Twelve replicates of 100 copies, 10 copies and 1 copy per well of the template DNA were subjected to 50 cycles of

PCR. The real-time amplification curves and dissociation are shown in Figure 2. The amplification curves show that

all the 100 and 10 copies per well samples amplified, with the 100 copies per well replicates all at very similar Cq

values. Six of the 1 copy per well replicates amplified and the other six were negative, which is to be expected on

the chance that a single copy will be present or absent in the well. Melting analysis of all the single copy replicates

which did amplify indicated that these all had the same Tm as the positives in higher copy number wells.

Some of the no template control (NTC) samples also showed some amplification. On studying the dissociation

curves, the Tm of the peaks shown by the NTC products were either around 81.5°C or 89.4°C compared to 86.8°C

of the positive samples. Therefore the amplification in the NTC samples was not contamination but rather some

unidentified non-specific product(s) which were not amplified in any of the other samples.

Figure 2: Amplification and dissociation curves for each of the Lambda DNA PCRs. The amplification curves are shown from

cycle 20. Red = 100 copies/well; Green = 10 copies/well; Yellow = 1 copy/well; Blue = no template controls (NTC). The

dissociation curves show product melting at 86.8°C together with non-specific products in the NTCs at 81.5°C or 89.4°C.

To clarify the real-time fluorescence results, the samples were run on agarose gels to visualise the PCR products

and these are shown in Figure 3.

Figure 3: Gel analysis of the PCR products. Each gel shows the 12 replicates for each of the four master mixes containing none

(NTC), 1 copy/well, 10 copies/well or 100 copies/well.



The banding pattern corresponded exactly with the fluorescence amplification curves. The two NTC samples which

gave a product with a high Tm, A1 and A4, gave a band which appeared on the gel to be slightly larger in size than

the bands from the positive samples. Without further analysis it is uncertain what this product is. The very low

molecular weight bands in the other NTCs corresponded to the samples which gave the product with the low Tm

value. These are most likely to be non-specific primer-dimer products.

To determine the certainty with which it was possible to distinguish replicates containing a single copy of the

Lambda DNA from replicates containing higher numbers of copies, the average and SDs of the Cq values for each

replicate group were determined and are shown in Figure 4.

A Student’s t-test was performed to determine the significance between the results. The t-test compares the actual

difference between the means of two sets of data in relation to the variation in the data expressed as the standard

deviation of the difference between the means (2). Calculated results are shown in Table 4.

Copies per well Average Cq SD t (9), p t (16), p t (22), p

0 42.16 3.03 6.31, <0.001

1 34.07 0.85 7.18, <0.001

10 31.47 0.66 17.8, <0.001

100 27.87 0.25

Table 4: Average Cq and SD for each of the replicate groups. A Student’s t-test (2, 3) performed on neighbouring replicate

groups indicates that the differences between the paired sets of samples are "very highly significant" with probability, p (of the

difference being due to chance) <0.001 for each pair tested.

The results indicate that the average Cq values from each replicate group are significantly different from each

other.

Conclusions

The determination of initial copy number was based on a sensitive fluorimetric DNA assay using a purchased

standard DNA and the copy number calculated using the molecular weight of the Lambda DNA template. The

results reflect what would be expected by diluting this DNA down to a theoretical single copy per reaction.

Amplification of low copy numbers generally involves increasing the number of cycling steps and this, together

with the lack of competition from target template in the reaction can increase the chance that products will be

amplified in the NTC samples. Tm analysis can help to identify whether any NTC products are due to

contamination, primer dimer amplification or non-specific amplification.

25

30

35

40

45

50

0 1 10 100

Cq

va

lue

Template copies per well

Average Cq

Figure 4: Average Cq data for each replicate

group plotted on a bar graph showing 1 SD

error bars.



The limit of detection (LOD) of qPCR assays is often determined by stochastic effects such as reaction efficiency and

whether or not non-specific products are formed. The LOD is defined as the lowest concentration at which 95% of

the positive samples are detected (4). In the tests performed here, the LOD would be below 10 copies per reaction

since 100% of the 10 copies per well samples amplified. The lowest theoretical LOD per PCR is 3 copies; this

assumes a Poisson distribution of positive and negative results, a 95% chance of including at least 1 copy in the PCR

and single-copy detection (4). The Poisson distribution predicts that in a large number of replicates containing an

average of one copy of starting template, about 37% should actually have no copies, about 37% should contain one

copy and about 18% should contain two copies (5). In the assay described in this application note, 50% of the single

copy per well reactions amplified which may indicate slightly more than 1 copy per well on average.

References (1) http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome

(2) http://archive.bio.ed.ac.uk/jdeacon/statistics/tress4a.html

(3) http://www.physics.csbsju.edu/stats/t-test.html

(4) Bustin SA, Benes V, et al. (2009) The MIQE Guidelines: minimum information for publication of quantitative

real-time PCR experiments. Clin Chem, 55(4): 611−622.

(5) http://www.invitrogen.com/etc/medialib/en/filelibrary/Nucleic-Acid-Amplification-Expression-

Profiling/PDFs.Par.70657.File.dat/Understanding%20Ct%20Application%20Note.pdf.

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application note a08-005a detecting low copy numbers

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