Empcr amplification method manual - libc - a

Figure 2: Schematic view of an assembled set up for vacuum-assisted emulsion breaking and bead recovery 3. Turn on the vacuum and aspirate the emulsions A and B from all the wells and collect them in the 50 ml tube, using a slow circular motion of the transpette tips at the bottom of the wells.

After aspirating all the emulsions, turn the transpette upside-down to help drain as much material as possible into the collection tube. Aspirate the rinse and turn the transpette upside-down to retrieve as much material as possible. SLOWLY aspirate an additional approximate 5 ml of isopropanol to collect any beads that may remain in the tubing. Turn off the vacuum, and remove and cap the 50 ml tube containing the amplified DNA beads. Take the 50 ml tube out of the hood. Vortex the 50 ml tube of collected emulsions.

Add isopropanol to a final volume of 35 ml and vortex to resuspend the pellet. Pellet the beads in a centrifuge at x g for 5 min RPM for the Eppendorf centrifuge, rotor F and carefully pour out the supernatant. Add 10 ml of Enhancing Buffer and thoroughly vortex to resuspend the pellet it is important to properly rinse the beads. Use glass rod or a spatula to break the aggregates, if necessary.

Add isopropanol to a final volume of 40 ml and vortex well. Pellet the beads in a centrifuge at x g for 5 min and carefully remove the supernatant.

Add isopropanol to a final volume of 35 ml of and vortex well. Add ethanol to a final volume of 35 ml of and vortex well. Add Enhancing Buffer to a final volume of 35 ml of and vortex well. Pellet the beads in a centrifuge at x g for 5 min and carefully remove the supernatant, leaving approximately 2 ml of Enhancing Buffer.

Spin-rotate-spin and discard the supernatant. Rinse the 50 ml tube with 1 ml of Enhancing Buffer, and add this rinse to the 1. Thoroughly rinse the bead pellet twice with 1 ml of Enhancing Buffer. Read the Material Safety Data Sheet for handling precautions. Add 1 ml of Melt Solution to the 1.

Incubate for 2 minutes at room temperature. Repeat Step 3 once. Add 1 ml of Annealing Buffer to the 1. Repeat Step 5 twice. Add 1 ml of Enhancing Buffer to the 1. Repeat Step 9 two more times. Set the tube aside at room temperature until Section 3. Vortex the tube of brown Enrichment Beads for 1 minute to resuspend its contents completely.

Discard the supernatant, taking care not to draw off any Enrichment Beads. Repeat Steps 4 to 6 once. After discarding the supernatant, remove the tube from the MPC. Rotate the tube on the LabQuake, at room temperature for 5 minutes. Invert the MPC several times and wait for the beads to pellet.

Wash the beads with Enhancing Buffer until there are no visible white beads remaining in the supernatant, as follows: a. Add 1 ml of Enhancing Buffer to the tube. Remove the tube from the MPC and vortex well. Place the tube back into the MPC to pellet the beads on the wall of the tube with the magnet.

Invert the MPC and wait for the beads to pellet. Repeat 6 to 10 times until white DNA beads are no longer being aspirated.

Optionally, collect the supernatant and spin to monitor when washes are complete. Vortex for 5 seconds, and place the enrichment tube in the MPC until the Enrichment Beads have pelleted. Transfer the supernatant containing the enriched DNA beads to a new 1. PCR technique was developed by Kary mullis in In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences primers. PCR - Science method.

Join Co-production practitioners network. Sign Up or Sign In. Powered by. Badges Report an Issue Terms of Service. Co-production practitioners network A network for co-production practitioners. For example, the sample may be collected for about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours.

In further embodiments, the method includes the detection of small DNA fragments. As described herein, the method results in an increase in sensitivity for DNA detection by a factor of up to one hundred or several hundred. In one embodiment, the DNA analysis results in an increase in signal over standard mutations assays, e. In one embodiment, the method may provide a greater than fold increase in signal over standard assays.

DNA found in urine consists of both transrenal and nontransrenal fractions and the amounts of non transrenal DNA extracted from large sample volumes can overwhelm sensitive assays intended to quantify mutations in transrenal DNA. Various size selection methods can be employed and are well known to those having ordinary skill in the art. In one embodiment, the urine sample is collected in a urine collection container.

In another embodiment, the DNA is extracted using a size selectivity method. In another embodiment, the size selectivity method is a membrane with a pore size that allows DNA to pass through ranging in size up to about 60 bp in length or up to about bp in length. For example, the pore size may allow DNA to pass through in a size of up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or bp in length.

In one embodiment, the pore size may allow DNA to pass through in a size of up to about , , , , , , , , , , , , , , or bp in length or similar. For example, the pore size may be 0. In one embodiment, the pore size is selected from 5 nm, 10 nm, 12 nm, 35 nm, 0.

The pore size used will depend on the size of the DNA molecules desired in the sample. In various embodiments, the desired size of the DNA molecule for analysis are about , , , , , , , , , , , , , or bp. In one embodiment, the DNA molecules for analysis may be less than bp in length. For example, the DNA molecules for analysis may be less than bp, less than 75 bp, less than 60 bp, or less than 50 bp in length.

In one embodiment, a method is developed to extract DNA from large volumes of urine suitable for downstream mutation analysis. As herein described, a three-pronged approach may be employed to large volume urine ctDNA sample processing and analysis, consisting of urine crossflow diafiltration, neutralization of PCR inhibitors, and removal of non-transrenal DNA.

In another embodiment, the sample collection can take place over a period of time greater than eight 8 hours and less than twenty-five 25 hours.

In other embodiments, the sample of urine from a patient may range from greater than 1 L to 4 L of urine. In another embodiment, the urine sample may be between 0. In another embodiment, the urine sample is greater than 0. In one embodiment, crossflow diafiltration combines dialysis for removal of soluble PCR inhibitors with concentration of urine prior to ctDNA extraction. Useful polyethersulfone PES membrane pore sizes are described herein to maximize recovery of, e.

In another embodiment the membrane to separate the ctDNA from the rest of the fluid is comprised of a hydrophilic membrane. In another embodiment, the membrane is a hydrophilic membrane. In another embodiment, the extracted ctDNA desired length is between 25 and 75 bp. In another embodiment the extracted ctDNA is at least 25 bp in length.

In another embodiment, the extracted ctDNA is greater than bp in length. In another embodiment, the desired extracted ctDNA is between 26 and 64 bp in length.

Moreover, alterations in crossflow diafiltration parameters, such as addition of significant amounts of a salt-less solution, can further improve DNA desalting.

In one embodiment, the salt-less solution is deionized water. In one embodiment the amount of deionized water added to the urine sample is a ratio of urine to deionized water.

In another embodiment the urine sample is diluted with up to 6 L of deionized water. For example, the urine may be diluted ata ratio of 0. To address this, PCR additives, e. In one embodiment, the method is used to extract DNA and detect a disease. In some embodiments, the cancer comprises a primary tumor.

In yet other embodiments, the cancer comprises non-metastatic tumor cells. In yet other embodiments, the cancer comprises metastatic tumor cells. In one embodiment, detecting the ctDNA in the sample comprises quantifying the copy number of a gene in the ctDNA sample. In some embodiments, the gene copy number is quantified per ml of sample. The methods described herein can be used to detect or identify specific nucleic acid sequences in a DNA sample.

Techniques for isolation of DNA are well-known in the art. Methods for isolating DNA are described in Sambrook et al. In various embodiments, the mutation is indicative of a disease, e. In one embodiment, the ctDNA may be analyzed for the presence of a specific mutant allele fraction, a genetic marker, a biomarker, a tumor marker, or a tumor recurrence marker. In one embodiment, the nucleic acid may be detected using conventional polymerase chain reaction PCR methods.

When performing conventional PCR, the final concentration of template is proportional to the starting copy number and the number of amplification cycles. In one embodiment, a given number of reactions is performed on a single sample and the result is an analysis of fragment sizes or, for quantitative real-time PCR qPCR , the analysis is an estimate of the concentration of the target sequences in the reaction-based on the number of cycles required to reach a quantification cycle Cq.

For qPCR methods, a fluorescent reporter dye is used as an indirect measure of the amount of nucleic acid present during each amplification cycle. The increase in fluorescent signal is directly proportional to the quantity of exponentially accumulating PCR product molecules amplicons produced during the repeating phases of the reaction. Reporter molecules may be categorized as; double-stranded DNA dsDNA binding dyes, dyes conjugated to primers, or additional dye-conjugated oligonucleotides, referred to as probes.

As the PCR progresses and the quantity of dsDNA increases, more dye binds to the amplicons and hence, the signal intensity increases. Alternatively, a probe or combination of two depending on the detection chemistry can add a level of detection specificity beyond the dsDNA-binding dye, since it binds to a specific region of the template that is located between the primers.

For digital PCR dPCR , the sample can be diluted and separated into a large number of reaction chambers or partitions.

In some embodiments, the partition may contain one or more copies of the target DNA. In some embodiments, the partition may contain two or more copies of the target DNA. The number of reaction chambers or partitions varies between systems, from several thousand to millions. The PCR is then performed in each partition and the amplicon detected using a fluorescent label such that the collected data are a series of positive and negative results.

For example, a sample is fractionated into thousands of droplets e. The droplets for use in ddPCR are typically nanoliter-sized droplets. As described herein, for methods employing ddPCR, the sample s may be partitioned into 20, nanoliter-sized droplets. This partitioning allows the measurement of thousands of independent amplification events within a single sample.

Digital PCR is an end-point PCR method that is used for absolute quantification and for analysis of minority sequences against a background of similar majority sequences, e. When using this technique, the sample is taken to limiting dilution and the number of positive and negative reactions is used to determine a precise measurement of target concentration. In an alternative format, the reactions may be run on integrated fluidic circuits chips.

These chips have integrated chambers and valves for partitioning samples and reaction reagents e. In one embodiment, a method is described for detection of mutations in very small DNA fragments. For example, two PCR cycles or more using extension primers may be employed to elongate a fragment of interest while also limiting PCR-errors.

In one embodiment, very short DNA fragments are targeted e. For example, the primers may have an overlap with the DNA molecule template that is 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29 bp, 30 bp, or more. Given that the exact DNA fragment sizes harboring a mutation of interest may be unknown in a clinical sample, extension primers are designed using the gene's native DNA sequence to extend a wide range of short DNA fragments.

PCR parameters including primer concentration, concentration of PCR additives, PCR annealing temperatures, and temperature ramp speeds are analyzed for contribution to maximizing elongation efficiency. The basic principle of emPCR is dilution and compartmentalization of template molecules in water droplets in a water-in-oil emulsion. Ideally, the dilution is to a degree where each droplet contains a single template molecule and functions as a micro-PCR reaction.

Elongation efficiency and false positive mutation rates may be analyzed to determine optimal PCR conditions, utilizing in vitro systems of varying mutant and wildtype DNA fragment sizes and ratios, and modeling human urine, which contains DNA of differing fragment lengths.

In one embodiment, cells may be ruptured by using a detergent or a solvent, such as phenolchloroform. In another embodiment, cells remain intact and cell-free DNA may be extracted. DNA may be separated from other components in the sample by physical methods including, but not limited to, centrifugation, pressure techniques, or by using a substance with affinity for DNA, such as, for example, silica beads.

After sufficient washing, the isolated DNA may be suspended in either water or a buffer. In illustrative embodiments, the primers and probes may be double-stranded or single-stranded, but the primers and probes are typically single-stranded. The primers and probes described herein are capable of specific hybridization, under appropriate hybridization conditions e. The primers and probes described herein may be designed based on having a melting temperature within a certain range, and substantial complementarity to the target DNA.

Methods for the design of primers and probes are described in Sambrook et al. Also within the scope of the invention are nucleic acids complementary to the probes and primers described herein, and those that hybridize to the nucleic acids described herein or those that hybridize to their complements under highly stringent conditions. Conditions for low stringency and moderately stringent hybridization are described in Sambrook et al.

In some illustrative aspects, hybridization occurs along the full-length of the nucleic acid. A sequence database can be searched using the nucleic acid sequence of interest.

In some embodiments, the percent identity can be determined along the full-length of the nucleic acid. Techniques for synthesizing the probes and primers described herein are well-known in the art and include chemical syntheses and recombinant methods.

Such techniques are described in Sambrook et al. Primers and probes can also be made commercially e. Techniques for purifying or isolating the probes and primers described herein are well-known in the art. The primers and probes described herein can be analyzed by techniques known in the art, such as restriction enzyme analysis or sequencing, to determine if the sequence of the primers and probes is correct.

The following examples are exemplary embodiments of the disclosure. One of ordinary skill in the art will understand that slight variations or substitutions may be made to achieve the same results. Those slight variations and substitutions are considered a part of the disclosure herein. Analysis of large volumes of urine is challenging because of the lack of appropriate commercial DNA extraction solutions. As DNA extraction introduces small amounts of PCR inhibitors one cannot simply subdivide a large urine sample into small ones and use routine extraction methods.

The data suggest that crossflow or tangential flow diafiltration using polyethersulfone PES membranes e. Fragment length distribution analysis of fetal transrenal DNA suggests that there are 10 to times more transrenal DNA molecules of base pair bp length than there are of those of bp length.

However, mutations in small e. The potential signal gain that can be achieved by quantifying mutations in DNA fragments bp can be estimated as the ratio of the amount of ctDNA from 30 to 60 bp in length over the amount of longer ctDNAs. Based on fetal transrenal DNA fragment length distribution, possible signal gain factors of far over are likely, suggesting that mutation detection across a range of small transrenal DNA fragments could dramatically increase sensitivity of ctDNA assays even independent from large volume urine DNA recovery.

Briefly, two PCR cycles using extension primers, each bp long are sufficient to elongate fragments of interest while also limiting PCR-errors. Targeting very short bp DNA fragments with both primers having 15 bp or more overlap with the DNA template allows assay design extension primers centered on mutation flanking the ddPCR probe site and specific amplification for virtually all single nucleotide alterations SNAs.

Given that the exact DNA fragment sizes harboring a mutation of interest are unknown in a clinical sample, extension primers are designed using the gene's native DNA sequence to extend a wide range of short DNA fragments. This approach will not limit analysis to a specific DNA fragment length. Fragments of interest with significantly less than 15 bp overlap cannot be extended with this method.

The capability to extract DNA from large volumes of urine suitable for downstream mutation analysis was developed. No solutions for DNA extraction from very large volumes of urine suitable for routine rare mutation analysis methods previously existed.

A scalable method was developed. To clearly demonstrate the signal gain associated with the analysis of large urine volumes over small volume urine analysis, identical DNA quantification assays i. DNA extracted from a hour urine samples using the optimized protocol as herein described produced about a fold higher ctDNA signal than DNA from about a 30 mL urine sample.

The data show that large volume urine-based ctDNA detection is feasible and identifies more mutant copies than small urine sample ctDNA analysis. The following inclusion criteria was used for all patients: i informed consent, ii non-genitourinary solid tumor with pathologically confirmed cancer diagnosis, iii advanced disease with an estimated 20 mL or greater of total tumor volume.

Feasibility: Patients with typically widely metastatic disease commonly will have clinical mutation testing data available, which was used to identify patient-specific mutations for assay development. Most patients will be recruited when they present for evaluation for palliative radiation therapy. There is a highly functional departmental clinical research infrastructure in place for recruitment and sample collections. Similar to very common hour urine laboratory tests performed for various reasons e.