We would like to acknowledge the contributions of all colleagues and collaborators to our original studies, whose results have been referred to in this manuscript (for illustrative purposes). The authors&#39; work is supported by grants from the National Institute of Dental and Craniofacial Research of the National Institutes of Health (1R01DE026043) to AB and from the University of California Tobacco-Related Disease Research Program to AB (TRDRP-26IR-0015) and ST (TRDRP-25IP-0001). The sponsors of the study had no role in study design, data collection, data analysis, data interpretation, writing of the report, or in the decision to submit for publication.

1. Genomic DNA Isolation from Mouse Embryonic Fibroblasts
NOTE: Primary mouse embryonic fibroblasts are isolated from embryos derived from BB transgenic mice with C57BL/6 genetic background, according to the published protocol53. The starting material for this protocol consists of 1 x 106 to 1 x 107 embryonic fibroblast cells treated with a test compound versus control. The harvesting and counting of these cells using standard methods are described in references10,54,55.
<ol>
	<li>Prepare buffer A (0.3 M Sucrose, 60 mM KCl, 15 mM NaCl, 60 mM Tris-HCl, pH 8.0, 0.5 mM spermidine, 0.15 mM spermine, and 2 mM EDTA), buffer B (150 mM NaCl and 5 mM EDTA, pH 7.8), and buffer C (20 mM Tris-HCl, pH 8.0, 20 mM NaCl, 20 mM EDTA, and 1% SDS) in advance, and preserve at room temperature (RT) for up to one year54,55.</li>
	<li>Resuspend cells in 2&#8211;4 mL buffer A in a 15 mL conical centrifuge tube by repeatedly pipetting up and down using a wide bore 1,000 &#181;L pipette tip.</li>
	<li>Add one volume (2&#8211;4 mL) buffer A containing 1% octylphenoxypolyethoxyethanol (e.g., Nonidet P40) using a glass serological pipette.</li>
	<li>Incubate the tube on ice for 5 min.</li>
	<li>Centrifuge the tube at 1,000 x g for 5 min at RT.</li>
	<li>Discard the supernatant and wash the pellet with 10&#8211;15 mL buffer A using a glass serological pipette.</li>
	<li>Resuspend the pellet in 2&#8211;4 mL buffer B.</li>
	<li>Add one volume (2&#8211;4 mL) buffer C containing 600 &#181;g/mL proteinase K.</li>
	<li>Incubate the tube for 3 h at 37 &#176;C.</li>
	<li>Add RNase A to a final concentration of 100 &#181;g/mL.</li>
	<li>Incubate the tube for an additional 1 h at 37 &#176;C.</li>
	<li>Add one volume (2&#8211;4 mL) phenol (saturated in 0.1 M Tris-HCl, pH 8.0):chloroform:isoamyl alcohol (25:24:1 vol/vol). 
	NOTE: Phenol can pose a severe health hazard and must be handled with extreme caution. Phenol is highly corrosive to the skin and readily absorbed through it, and exhibits other effects. When handling phenol, always use double gloving, wear protective eyewear, and work in a chemical fume hood.</li>
	<li>Mix well by inverting the tube for 5 min on a tube rotator at 4 &#176;C.</li>
	<li>Centrifuge the tube at 1,000 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the aqueous phase (top layer) to a new tube using a glass serological pipette.</li>
	<li>Repeat phenol:chloroform:isoamyl alcohol extraction 1 to 3 times until the aqueous phase is clear and the interface is no longer turbid.</li>
	<li>Add 1/10 volume (200&#8211;400 &#181;L) 3M Sodium acetate, pH 5.2.</li>
	<li>Add 2.5 volume (5&#8211;10 mL) 100% ethanol (chilled) and invert the tube gently by hand for 2&#8211;3 min.</li>
	<li>Spool the DNA with a glass hook and transfer it to a new tube containing 1&#8211;5 mL 70% Ethanol and wash it thoroughly. Alternatively, centrifuge the tube at high speed (3,500 x g) at 4 &#176;C to pellet the DNA and wash it thoroughly with 1&#8211;5 mL 70% Ethanol.</li>
	<li>Centrifuge the tube at high speed (3,500 x g) at RT, remove the supernatant, and air-dry the DNA for 10-15 min.</li>
	<li>Dissolve the DNA in 10&#8211;100 &#181;L TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), and store at 4 &#176;C (for short storage of up to one month) or at -20 &#176;C (for longer storage period). The optimal concentration for DNA for the &#955; Select cII assay is 0.5&#8211;1.0 &#181;g/&#181;L (in TE buffer)56.</li>
</ol>
2. In Vitro Packaging Reaction
<ol>
	<li>Per one packaging reaction, add ~5 &#181;g genomic DNA (final volume: 8&#8211;12 &#181;L, genomic DNA was isolated in Section 1 from mouse embryonic fibroblasts treated in vitro with a test compound or control) to a microcentrifuge tube containing the first reaction mix (~10 &#181;L). 
	NOTE: In-house prepared or commercially available &#955; packaging extracts are used in different laboratories. In the commercial packaging extract kit used here56 (see the Table of Materials), red tubes contained the first reaction mix (~10 &#181;L) and blue tubes contained the second reaction mix (~70 &#181;L for at least 5 reactions).</li>
	<li>Incubate the tube for 90 min at 30 &#176;C.</li>
	<li>Add the required volume (~12 &#181;L) of the second reaction mix to the tube.</li>
	<li>Incubate the tube for an additional 90 min at 30 &#176;C.</li>
	<li>Add 1.1 mL of SM buffer to the tube. 
	NOTE: 1 mL of this solution (i.e., packaging reaction mixture) will be used for screening &#955; cII-mutants. The remainder will be used for titering.
	<ol>
		<li>Prepare SM buffer by mixing 5.8 g NaCl, 2.0 g MgSO4.7H2O, 50 mL 1 M Tris-HCl (pH 7.5), and 5 mL 2% (w/v) gelatin. Add dH2O to a final volume of 1 liter, autoclave for 30 min. Store at room temperature for up to 1 year.</li>
	</ol>
	</li>
	<li>Vortex the tube containing the packaged DNA sample for 10 s at RT (vigorous vortexing).</li>
	<li>Pulse spin the tube in a microcentrifuge and store on ice until ready to use. If the sample is not going to be used within the same day of packaging, add 50 &#181;L of chloroform per mL of packaged DNA sample, vortex gently, and store at 4 &#176;C for up to 2 weeks.</li>
</ol>
3. Preparing the E. coli G1250 Bacterial Culture
<ol>
	<li>At least two days before plating, make a few bacterial streak plates from Escherichia coli (E. coli) G1250 on TB1-kanamycin agar plates.
	<ol>
		<li>Prepare TB1-kanamycin agar plates as follows. Mix 5.0 g NaCl, 10.0 g Casein peptone, and 12.0 g agar. Add 800 mL dH2O. Add 1 mL 0.1% Thiamine hydrochloride. Adjust pH to 7.0 with NaOH or HCl. Add dH2O to a final volume of 1 liter.</li>
		<li>Mix well and autoclave for 30 min. Allow to cool to 55 &#176;C. Add 50.0 mg Kanamycin and mix. Pour into sterile 100-mm petri dishes (20&#8211;25 mL per dish). Store plates at 4 &#176;C for up to two weeks.</li>
	</ol>
	</li>
	<li>Incubate the bacterial streak plates in a stationary 30 &#176;C incubator for at least 24 h.</li>
	<li>One day before plating, combine 10 mL of TB1 liquid medium with 100 &#181;L of 20% (w/v) maltose-1 M MgSO4 solution in a sterile 50 mL screw-cap conical tube.
	<ol>
		<li>Prepare TB1 liquid medium as follows. Mix 5.0 g NaCl and 10.0 g Casein peptone, then add 800 mL dH2O, 1 mL 0.1% Thiamine hydrochloride, and adjust the pH to 7.0 with NaOH or HCl. Then add dH2O to a final volume of 1 liter, mix well, and autoclave for 30 min. Store the medium at RT for up to three months.</li>
		<li>Prepare 20% (w/v) Maltose-1 M MgSO4 as follows. Mix 20.0 g Maltose and 24.6 g MgSO4. 7H2O, then add dH2O to a final volume of 100 mL. Filter sterilize and store at 4 &#176;C for up to 6 months.</li>
	</ol>
	</li>
	<li>Inoculate the liquid medium with several colonies from the bacterial streak plate using a sterile inoculating loop56.</li>
	<li>Incubate the liquid culture overnight in a 30 &#176;C shaking incubator with vigorous shaking (250&#8211;300 rpm).</li>
	<li>On the day of plating, centrifuge the conical tube containing the G1250 liquid culture at 1,500 x g for 10 min at RT to pellet the bacterial cells.</li>
	<li>Discard the supernatant and resuspend the cell pellet in 10 mL of 10 mM MgSO4.</li>
	<li>Measure the absorbance of a 1:10 dilution of the cell suspension at wavelength 600 nm using a UV-Vis spectrophotometer (e.g., 100 &#181;L cell suspension + 900 &#181;L 10 mM MgSO4)56.</li>
	<li>Dilute the cell suspension to a final OD600 of 0.5 with 10 mM MgSO4. The prepared suspension of G1250 E. coli with OD600 = 0.5 is referred to as &#39;the G1250 plating culture&#39;.</li>
	<li>Store the G1250 plating culture on ice and use within 1-2 h.</li>
</ol>
4. Plating the Packaged DNA Samples
<ol>
	<li>Per one packaged DNA sample, prepare sixteen sterile 14 x 100 mm2 round-bottom tubes and sixteen TB1 agar plates. Ten from each set will be used for screening and six for titering (three for Titer 20 and three for Titer 100).
	<ol>
		<li>Prepare TB1 agar plates as follows. Mix 5.0 g NaCl, 10.0 g Casein peptone, and 12.0 g Agar, then add 800 mL dH2O, and 1 mL 0.1% Thiamine hydrochloride. Adjust the pH to 7.0 with NaOH or HCl, and add dH2O to a final volume of 1 L.</li>
		<li>Mix well and autoclave for 30 min. Allow the mixture to cool to 55 &#176;C, then pour it into sterile 100 mm Petri dishes (20&#8211;25 mL per dish). Store the plates at 4 &#176;C for up to two weeks. 
		NOTE: TB1 agar plates should be prepared at least 24 h prior to use.</li>
	</ol>
	</li>
	<li>Aliquot 200 &#181;L of the G1250 plating culture into each round-bottom tube.</li>
	<li>For titering, make a 1:100 dilution of the packaged DNA sample and mix well by vortexing (i.e., 10 &#181;L packaged DNA sample + 990 &#181;L SM buffer).</li>
	<li>Add 20 &#181;L of the 1:100 dilution to each of the three Titer 20 tubes.</li>
	<li>Add 100 &#181;L of the 1:100 dilution to each of the three Titer 100 tubes.</li>
	<li>For screening, add 100 &#181;L of the &#39;undiluted&#39; packaged DNA sample to each of the ten screening tubes.</li>
	<li>Process all titering and screening tubes (2 x 3 + 10 = 16 tubes), as follows: mix well by vortexing for approximately 10 s, and then incubate at room temperature for 30 min to allow the host E. coli to adsorb the phages.</li>
	<li>Add 2.5 mL microwaved molten TB1 top agar (cooled to 55 &#176;C) to each titer or screening tube, mix immediately by vortexing (gently), and pouring into the appropriate titer or screening TB1 agar plates.
	<ol>
		<li>Prepare TB1 top agar as follows. Mix 5.0 g NaCl, 10.0 g Casein peptone, and 7.0 g Agar, then add 800 mL dH2O. Add 1 mL 0.1% Thiamine hydrochloride, then adjust the pH to 7.0 with NaOH or HCl. Finally, add dH2O to a final volume of 1 liter.</li>
		<li>Mix well and autoclave for 20&#8211;25 min. Store at room temperature for up to three months.</li>
		<li>Prior to use, melt the prepared TB1 top agar in a microwave, mix well, and allow to cool to 55 &#176;C in a water bath. 
		NOTE: The molten and cooled TB1 top is added to the contents of each titer or screening tube, and after mixing, is poured into the appropriate titer or screening TB1 agar plates.</li>
	</ol>
	</li>
	<li>Let the plates stand for 15&#8211;30 min at room temperature, with the lid ajar to prevent condensation.</li>
	<li>Invert the plates and place the ten screening plates in a stationary 24 &#176;C incubator and incubate for 46&#8211;48 h (i.e., selective conditions) and the six titer plates in a stationary 37 &#176;C incubator and incubate for 24 h/overnight (i.e., non-selective conditions). 
	NOTE: For quality assurance and standardization of results, commercially available control phage solutions containing a mixture of wildtype and mutant cII with known mutant frequency are plated alongside the packaged DNA samples and included in all assay runs56.</li>
</ol>
5. Examining the Titer and Screening Plates to Determine the cII Mutant Frequency
<ol>
	<li>Following the 24 h/overnight incubation at 37 &#176;C, count the number of plaques formed in each of the three Titer 20 plates and Titer 100 plates. To more easily identify the plaques, hold the plate next to a white light box and against a dark background with lid removed (see Figure 2).</li>
	<li>Make an average (mean) of the number of plaques counted in each set of Titer 20 plates and Titer 100 plates (triplicate, each).</li>
	<li>Choose the average number from the set of titer plates that falls closest to the range of 50&#8211;200 plaques per plate set.</li>
	<li>Calculate the total number of plaques screened in the ten screening plates as follows: 
	Total plaques screened = (Mean number of plaques in the chosen set of titer plates &#247; Number of &#181;L of dilution used per chosen titer plate) x Dilution factor x Number of &#181;L of packaged DNA sample per screening plate [100 &#181;L/plate] x Number of screening plates [10]. 
	NOTE: For example, if the mean number of plaques per plate in the set of Titer 20 plates is 128 and the corresponding number in the set of Titer 100 plates is 478, the number 128 will be used for calculation of the total number of plaques screened, as follows: 
	(128 &#247; 20) x 100 [dilution factor] x 100 [&#181;L/plate] x 10 [plates] = 640,000 
	This is equivalent to multiplying the mean number of plaques by 5,000 or 1,000, if the average number is based on counts from Titer 20 plates or Titer 100 plates, respectively.</li>
	<li>Following 46&#8211;48 h incubation at 24 &#176;C, count the total number of plaques formed in the ten screening plates (follow the instructions provided in Section 5.1). 
	NOTE: The cII mutant frequency is calculated by dividing the total number of plaques formed in the screening plates by the total number of plaques screened. Using the above example, if the total number of plaques counted in the ten screening plates is 112, the cII mutant frequency for this DNA sample would be 112 &#247; 640,000 = 17.5 x 10-5.</li>
</ol>
6. Verification of the Putative &#955; cII Mutants, PCR Amplification, and DNA Sequencing
<ol>
	<li>Core the plaque in question with a sterile, wide-bore pipet tip and expel it (by pipetting up and down) into a sterile microcentrifuge tube containing 500 &#181;L of sterile SM buffer.</li>
	<li>Incubate for at least 2 h at room temperature, or at 4 &#176;C overnight, to allow the phage particles to elute from the agar plug.</li>
	<li>In a sterile 14 x 100 mm2 round-bottom tube, mix 200 &#181;L of the G1250 plating culture with 1 &#181;L of the cored phage solution, and incubate for 30 min at RT.</li>
	<li>Plate the sample using 2.5 mL of 55 &#176;C molten TB1 top agar and incubate the plate at 24 &#176;C for 46&#8211;48 h (selective conditions), as described in Sections 4.8&#8211;4.10.</li>
	<li>Once the secondary plaques have formed, use a pipette tip to carefully pick a single well-isolated verified &#955; cII-mutant plaque (avoid touching the lower agar). 
	NOTE: Replating the putative cII mutant plaques serves two purposes: (i) plating artefacts may sometimes be mistaken for small-size plaques; and (ii) an agarose core taken from a screening plate may contain non-mutant phage(s) together with a mutant phage. Secondary plaques from a low-density replating will provide an uncontaminated mutant template for PCR and subsequent DNA sequence analysis.</li>
	<li>Transfer the plaque to a microcentrifuge tube containing 25 &#181;L double-distilled water by pipetting up and down.</li>
	<li>Place the tube in boiling water for 5 min.</li>
	<li>Centrifuge at maximum speed (18,000 x g) for 3 minutes at RT.</li>
	<li>Transfer 10 &#181;L of the supernatant immediately to a new microcentrifuge tube containing 40 &#181;L of a PCR mastermix in which the final concentrations of the reagents are 1 x Taq PCR buffer, 10 pmol each of the forward and reverse primers, 12.5 nmol of each dNTP, and 2.5 U of Taq DNA polymerase. 
	NOTE: The forward and reverse primers are as follows: 5&#39;-CCACACCTATGGTGTATG-3&#39; (positions -68 to -50 relative to the cII start codon) and 5&#39;-CCTCTGCCGAAGTTGAGTAT-3&#39; (positions +345 to +365 relative to the cII start codon), respectively.</li>
	<li>Amplify the template using the following cycling parameters: a 3 min denaturation at 95 &#176;C, followed by 30 cycles of 30 s at 95 &#176;C, 1 min at 60 &#176;C, and 1 min at 72 &#176;C, with a final extension of 10 min at 72&#160;&#176;C.</li>
	<li>Purify the 432 bp PCR product containing the cII gene and flanking regions using commercially available PCR purification kits, according to the manufacturer&#39;s instructions.</li>
	<li>Perform DNA sequencing using an appropriate sequencing platform, and analyze the resulting DNA sequences to detect mutations in the cII transgene (see the note below). 
	NOTE: This is best achieved by alignment with the reference cII sequence using software, such as the web-based T-Coffee sequence alignment server. For instructions on how to use the program, visit: <a href="http://tcoffee.crg.cat/">http://tcoffee.crg.cat/</a></li>
</ol>

All the authors declare no conflict of interest.

The &#955; Select cII assay is used for detection of mutations in the cII transgene recovered from the genomic DNA of cells derived from organs/tissues of BB rodents3. The genome of these transgenic animals contains multiple tandem copies of the chromosomally integrated &#955;LIZ shuttle vector, which carries the cII (294 bp) and lacI (1,080 bp) transgenes, as the mutational reporter genes1,2,...

A wide range of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic Big Blue (referred to hereafter as BB) mice and the &#955; Select cII Mutation Detection System have been employed for mutagenicity experiments by this group and many other research groups worldwide1,2,3,4,5,6,7,8,9. For the past 16 years, we have investigated the mutagenic effects of various chemical and/or physical agents using these transgenic animals or their corresponding embryonic fibroblast cell cultures treated with a test compound, and subsequently analyzed the phenotype and genotype of the cII transgene by the &#955; Select cII assay and DNA sequencing, respectively10,11,12,13,14,15,16,17,18,19,20,21,22,23,24. The genome of these transgenic animals contains a bacteriophage &#955; shuttle vector (&#955;LIZ) integrated on chromosome 4 as a multi-copy head-to-tail concatemer1,2,25. The &#955;LIZ shuttle vector carries two mutational reporter genes, namely the lacI and cII transgenes1,2,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47. The &#955; Select cII assay is based on the recovery of the &#955;LIZ shuttle vectors from the genomic DNA of cells derived from organs/tissues of transgenic animals1,2,25. The recovered &#955;LIZ shuttle vectors are then packaged into &#955; phage heads capable of infecting an indicator host Escherichia coli. Subsequently, the infected bacteria are grown under selective conditions to allow for scoring and analysis of mutations in the cII transgene1,3.
Here, we describe a detailed protocol for the &#955; Select cII assay, which consists of isolation of genomic DNA from cells/organs of transgenic animals treated in vitro/in vivo with a test compound, retrieval of the &#955;LIZ shuttle vectors from the genomic DNA, packaging of the vectors into infectious &#955; phages, infection of the host E. coli with the bacteriophages, identification of the cII-mutants under selective conditions to determine the cII mutant frequency, and DNA sequence analysis to establish the cII mutation spectrum. The protocol can be applied to transgenic mouse/rat cell cultures treated in vitro with a chemical/physical agent of interest, or tissues/organs of the corresponding animals treated in vivo with the test chemical/agent1,2,4,48,49,50,51,52. A schematic presentation of the &#955; Select cII assay is shown in Figure 1.

<table><tbody><tr><td>Agar</td><td>MO Bio Laboratories, Inc.</td><td>12112-05</td><td>Bacteriological grade</td></tr><tr><td>BigDye Terminator v3.1 Cycle Sequencing Kit</td><td>Thermo Fisher Scientific</td><td>4337455</td><td>None</td></tr><tr><td>Casein Peptone</td><td>Alfa Aesar</td><td>H26557</td><td>None</td></tr><tr><td>Gelatine</td><td>J. T. Baker</td><td>2124-01</td><td>Powder</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP 229-1 / M-13750</td><td>None</td></tr><tr><td>LB Agar</td><td>Fisher Scientific</td><td>BP 9724-500</td><td>None</td></tr><tr><td>QIAquick PCR purification kit</td><td>Qiagen</td><td>8104</td><td>50 PCR purification reactions</td></tr><tr><td>Sodium Acetate Trihydrate</td><td>Fisher Scientific</td><td>M-15756</td><td>None</td></tr><tr><td>&lt;em&gt;Taq5000&lt;/em&gt; DNA Polymerase&amp;nbsp;</td><td>Qiagen</td><td>201207</td><td>None</td></tr><tr><td>Thiamine Hydrochloride</td><td>Macron Fine Chemicals</td><td>2722-57</td><td>None</td></tr><tr><td>Transpack Packaging Extract</td><td>Stratagene Corp., Acquired by BioReliance | Sigma-Aldrich Corp.</td><td>200223</td><td>50 packaging reactions</td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP 152-1 / EC 201-064-4</td><td>None</td></tr><tr><td>Trypton</td><td>Biosciences</td><td>RC-110</td><td>None</td></tr></tbody></table>

the lambda select cii mutation detection system

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (&#955;) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

Depending on data distribution, parametric or non-parametric tests are used to determine the significance of difference in the cII mutant frequency between treatment and control groups (i.e., induced versus spontaneous mutant frequencies). Comparison of the induced cII mutant frequencies across different treatment groups is made by various (pairwise) statistical tests, as applicable. The hypergeometric test of Adams and Skopek is commonly used to compare the ove...

Watch this Scientific Journal Video about The Lambda Select cII Mutation Detection System at JoVE.com

The Lambda Select cII Mutation Detection System

We describe a detailed protocol for the Lambda Select cII mutation assay in cultured cells of transgenic rodents or the corresponding animals treated with a chemical/physical agent of interest. This approach has been widely used for mutagenicity testing of carcinogens in mammalian cells.

The Lambda Select cII Mutation Detection System

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of ...

the-lambda-select-cii-mutation-detection-system

Research

JoVE Journal

Immunology and Infection

7.8K Views.  University of Southern California. We describe a detailed protocol for the Lambda Select cII mutation assay in cultured cells of transgenic rodents or the corresponding animals treated with a chemical/physical agent of interest. This approach has been widely used for mutagenicity testing of carcinogens in mammalian cells.

Video: The Lambda Select cII Mutation Detection System

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging resistance mutations during therapy, or when patients have few circulating tumor cells. Wild-type blocking PCR followed by sequencing analysis offers high sensitivity, flexibility, and simplicity as a methodology for detecting these low frequency mutations. By adding a custom designed locked nucleic acid oligonucleotide to a new or previously established conventional PCR based sequencing assay, sensitivities of approximately 1 mutant allele in a background of 1,000 WT alleles can be achieved (1:1,000). Sequencing artifacts associated with deamination events commonly found in formalin fixed paraffin embedded tissues can be partially remedied by the use of uracil DNA glycosylase during extraction steps. The optimized protocol here is specific for detecting MYD88 mutation, but can serve as a template to design any WTB-PCR assay. Advantages of the WTB-PCR assay over other commonly utilized assays for the detection of low frequency mutations including allele specific PCR and real-time quantitative PCR include fewer occurrences of false positives, greater flexibility and ease of implementation, and the ability to detect both known and unknown mutations.

Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging ...

Genetics

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

Cancer Research

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

Biochemistry

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicine

Naked-Eye Detection of Rare Point Mutations in DNA

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen application of the method is the identification of rare mutations in circulating cell-free DNA from liquid biopsies, with a relevance in cancer diagnostics and stratification of oncological patients for personalized therapy. As a proof of concept, the test has been designed to detect the BRAFV600E mutation in the BRAF gene, which is important to identify the sub-group of melanoma patients that can benefit from targeted therapies with BRAF inhibitors. However, this colorimetric test can be easily generalized to other somatic mutations of clinical relevance due to the use of universal detection probes, thus providing strong potential in oncological diagnostics.
The test detects 0.5% of BRAFV600E in an excess of BRAFWT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients. In contrast to commercial assays based on real-time PCR, this test requires minimal instrumentation and processing, as it can be performed on DNA amplified with a standard PCR (or isothermal techniques) and provides a naked-eye readout with a one-tube reaction of a few steps in only one hour. At present, the test has been used only on synthetic DNA samples. However, the latter have been designed to mimic a real sample amplified from circulating cell-free DNA, to favor the translation of the test to clinical diagnostics.

This protocol permits the naked-eye identification of point mutated DNA in a 200-fold excess of wild type DNA molecules, by exploiting gold nanoparticles and paramagnetic microparticles.

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen ...

The Lambda Select cII Mutation Detection System

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Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Naked-Eye Detection of Rare Point Mutations in DNA