HALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL. Version 1.0.3, April 2011 For research use only

Transcription

1 HALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL Version 1.0.3, April 2011 For research use only

2 HALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL Halo Genomics AB, 2011 No part of this protocol may be reproduced in any format without a written consent from Halo Genomics according to international copyright laws. Information and material contained in this document are provided as is and may be changed without notice in future editions. Edition version 1.0.3, April 2011 Printed in Sweden Halo Genomics Dag Hammarskjölds väg 54A SE Uppsala SWEDEN Technical support and advice is available at: or by sending an to: support@halogenomics.com The product and the material and information contained in this document are intended for research use only and should not be used for diagnostic purposes under any circumstances. Store all reagents at -20 C except for the Magnetic beads which are to be stored at +4 C. For expiry dates please refer to the outer packaging label. 1

3 Version 1.0.3, April For research use only TABLE OF CONTENTS Overview 3 HaloPlex PCR - Target Enrichment & Library Preparation Kit 3 HaloPlex PCR Technology 3 Equipment and reagents 5 Included reagents 5 Required equipment and reagents 6 Optional equipment and reagents 6 DNA sample guidelines 6 HaloPlex PCR protocol for enrichment 7 Phase I Restriction enzyme digestion 7 Step 1. Prepare ten DNA samples and one control 7 Step 2. Prepare the restriction enzymes 7 Step 3. Preparing the reaction plate: aliquoting the RE master mix strip 9 Step 4. Preparing the reaction plate: aliquoting the DNA sample mixes 9 Step 5. Incubate the reaction plate 10 Step 6. Pooling the restriction enzyme digestions 10 Phase II Hybridization to HaloPlex Probes 10 Step 1. Add the HaloPlex Probes 10 Step 2. Incubate over night 11 Phase III Solid phase capture and DNA ligation 11 Step 1. Prepare the Magnetic Beads 11 Step 2. Add the Magnetic Beads 12 Step 3. Wash the beads 12 Step 4. Ligate the target fragments 13 Phase IV Enrichment by PCR 13 Step 1. Haloase A treatment 13 Step 2. Haloase B treatment 14 Step 3. Amplify targeted fragments with PCR 15 Purify, validate and pool 16 Appendix 17 2

4 HALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL OVERVIEW HaloPlex PCR - Target Enrichment & Library Preparation Kit This document describes the practical procedures for using the HaloPlex PCR - Target Enrichment & Library Preparation Kit. The genomic regions to be enriched are defined individually by the customer prior to ordering the kit through the online Halo Design Wizard available at: Technology The HaloPlex PCR technology described in this protocol enriches target regions in a DNA sample in four phases (see figure 1): I. Restriction enzyme digestion, II. Hybridization to HaloPlex probes and introduction of Illumina sequence motifs, III. Solid phase capture and DNA ligation, and IV. PCR enrichment using indexed primers. In the first phase, each DNA sample is fragmented in eight parallel restriction digestion reactions. In phase II, the HaloPlex probes are added to the pooled digestion reactions and allowed to hybridize to targeted fragments over night. The Halo- Plex probes are designed to guide the circularization of target fragments and also contains the Illumina sequence motifs which are being incorporated during circularization. The biotin molecules attached to the HaloPlex probes enable capture of the probe-target complex on streptavidin coated magnetic beads in phase III. Following DNA ligation, the circles are amplified by PCR in step IV.This step amplifies the targeted fragments and associates them with the sequence motifs needed for Illumina sequencing. By using differently indexed primer pairs, samples can be pooled after this step. 3

5 Version 1.0.3, April For research use only I. DIGEST SAMPLE DNA II. HYBRIDIZE PROBES - SEQUENCING MOTIFS ARE INCORPORATED III. PURIFY AND LIGATE TARGETS IV. AMPLIFY TARGETED FRAGMENTS WITH PCR - SAMPLE BARCODES ARE INTRODUCED Sequence library ready for Emulsion/Bridge PCR Non-target DNA Target regions HaloPlex Probe 4

6 HALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL EQUIPMENT AND REAGENTS Included reagents -20 C Storage HaloPlex Probes ( ) Hybridization Solution ( ) Ligation Solution ( ) Wash Solution ( ) Capture Solution ( ) DNA Ligase ( ) RE Buffer ( ) Enzyme Strip 1 ( ) Enzyme Strip 2 ( ) RE Control DNA ( ) Haloase A Haloase A Buffer Haloase B Haloase B Buffer Primer Cassette +4 C Storage Magnetic Beads ( ) 5

7 Version 1.0.3, April For research use only Required equipment and reagents 96-well ring magnet plate for PCR μ-tubes (Agencourt, #A32782) Benchtop microcentrifuge for PCR μ-tubes (VWR, , or equivalent) Thermal cycler (ABI, # ) Qubit Fluorometer (Invitrogen, #Q32857) Quant-iT TM dsdna BR Assay Kit (Invitrogen, #Q32853) Qubit TM assay tubes (Invitrogen, #Q32856) 8-well PCR μ-strips with lids (Sarstedt, # , or equivalent) 96 well PCR plate with film (Abgene, # , or equivalent) Phusion Hot Start II High-Fidelity DNA Polymerase (Finnzymes, F-549L) Gel electrophoresis equipment Ampure XP (Agencourt, #A63880) Multiplexing Sample Preparation Oligonucleotide Kit (Illumina, #PE ) Optional equipment and reagents µl multi-channel pipette (Eppendorf, # , or equivalent) DNA sample guidelines Please make sure that the DNA samples are of high quality, meaning non-degraded with a A260/ A280 ratio of 1.8 to 2.0. Use the Qubit dsdna BR Assay to accurately determine the concentration of the DNA samples. 6

8 HALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL PHASE I - RESTRICTION ENZYME DIGESTION In the first phase, the DNA sample is aliquoted in eight different reactions, each containing two restriction enzymes. Restriction cleavage and pooling of the reactions results in a digested DNA sample containing both target and non-target regions of the genome. Step 1. Preparing ten DNA samples and one control In step 1, 900 ng of each of the ten DNA samples are diluted in purified H2O. As control of efficient digestion, the supplied RE Control DNA sample is prepared as well. 1.1 For each DNA sample, including the RE Control DNA ( ), create a DNA Sample mix by combining 900 ng DNA and purified H 2 0 to a total volume of 45 μl. Practical Note: preparing the DNA sample mixes in 0.3 ml PCR µ-strips, allows for easy dispensing using a multipipette in step µl per sample 900 ng DNA + ddh2o = 45 µl S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Ctrl DNA Sample mixes 1-10 RE Control DNA 1.2 Store the samples on ice until Step 4.1 on page 9. Step 2. Preparing the restriction enzymes In step 2, the master mixes for the eight enzyme reactions are prepared by combining two different enzymes to each well in an 8-well μ-strip. The restriction enzymes are mixed by adding enzyme A from Enzyme Strip 1 ( ) and enzyme A from Enzyme Strip 2 ( ) into well A of the RE master mix strip. Enzymes B-H are mixed in the same manner. The volumes stated in the restriction digestion protocol are for preparing mastermixes for digestion of 10 DNA samples and the RE Control DNA. To avoid pipetting errors, it is strongly recommended to not prepare less than 5 samples at a time. For each sample, the eight separate digestion reactions should contain: Reagent 1 rxn (µl) 11 rxns + 1 extra RE Buffer ( ) Enzyme from strip 1 ( ) Enzyme from strip 2 ( ) DNA sample mix 20 ng/µl Total volume: 10-7

9 Version 1.0.3, April For research use only! NOTE! It is CRITICAL that the pairs of enzymes are mixed together correctly in order for the reagent kit to work. Please pay careful attention to the instructions on how to mix the restriction enzymes. RE Buffer 2.1 Prepare a RE master mix strip by adding 48 μl RE Buffer ( ) to each tube of a fresh 8-well μ-strip. Label the eight tubes in the strip A to H. 48 µl per well A B C D E F G H RE master mix strip 2.2 Add 6 μl enzyme from tubes A to H from the Enzyme Strip 1 ( ) to each corresponding tube A to H in the RE master mix strip. Hint: keep the green labeled tube A to your left. 6 µl to corresponding well Enzyme Strip 1 A B C D E F G H A B C D E F G H RE master mix strip 2.3 Add 6 μl enzyme from tubes A to H from the Enzyme Strip 2 ( ) to each corresponding tube A to H in the RE master mix strip. Hint: keep the orange labeled tube A to your left. 6 µl to corresponding well Enzyme Strip 2 A B C D E F G H A B C D E F G H RE master mix strip 2.4 Keep the RE master mix strip on ice while preparing for the next step. 8

10 Empty Empty HALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL Step 3. Preparing the reaction plate: aliquoting the RE master mix strip In this step the RE master mix strip will be aliquoted into a 96 well reaction plate. In this format, each ROW contains a different combination of restriction enzymes. Column 12 on the reaction plate will be kept empty. 3.1 From the RE master mix strip (prepared in step 2), pipette 5 μl from tube A into all wells in row A of the 96 well reaction plate. 3.2 Continue by dispensing 5 μl RE master mix from tube B into all wells in row B of the 96 well reaction plate. RE master mix A-H A B C D E F G H 5 µl per well A B C D E F G H Continue in the same manner by filling up rows C through H with their respective restriction enzyme master mixes. From the RE master mix strip, pipette 5 µl from tube A into all wells in row A. Continue with 5 µl from tube B into wells in row B and so on. When this step is finished, each ROW of the 96 well plate should contain 5 μl per well of the same restriction enzyme combination. The experienced user is recommended to use a multipipette for this step in the procedure. Step 4. Preparing the reaction plate: aliquoting the DNA sample mixes In this step, the DNA sample mixtures prepared in step 1 will be aliquoted into the 96 well reaction plate prepared in Step 3. In this format, each COLUMN in the 96 well reaction plate digests one DNA sample in eight different enzyme reactions. Column 12 on the reaction plate will be kept empty. 4.1 To the 96 well reaction plate, add 5 μl of DNA sample mix 1 into each of the eight wells of column 1. Change tips after each pipetting step to avoid cross contamination of enzymes between wells. 4.2 From DNA sample mix 2 add 5 μl to each of the eight wells of column Continue with adding DNA sample mix 3 to column 3, sample 4 to column 4 and so on. 5 µl per well A B C D E F G H DNA Sample mixes 1-10 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 La Add 5 µl DNA sample mix 1 (S1) to each well in column 1, S2 to wells in column 2 and so on. RE Control DNA

11 Empty Version 1.0.3, April For research use only Step 5. Incubate the reaction plate 5.1 Incubate in a thermal cycler at 37 C for four hours followed by enzyme inactivation at 80 C for 20 min. The prepared 96 well plate should now contain restriction digestions of the genomics DNA samples. Each column comprises one DNA sample and each row comprises one restriction enzyme combination. Step 6. Pooling the restriction enzyme digestions 6.1 Following deactivation of the restriction enzyme reactions, pool the eight 10 μl reactions from the same sample (i.e. from the same column) into a 0.3 ml PCR tube and close the lid. Do not pool the RE Control DNA reactions. 6.2 Validate efficient digestion by running the RE Control DNA on a gel (see Appendix for protocol). A B C D E F G H The digested samples can be frozen for long-time storage. Once the hybridization reaction have started there are no more stopping points. PHASE II - HYBRIDIZATION TO HALOPLEX PROBES The HaloPlex probes are designed to hybridize selectively to fragments originating from the target region, guiding circularization of the targeted fragments. During this process Illumina sequence motifs are also incorporated. Step 1. Add the HaloPlex probes 1.1 Prepare the hybridization reaction in a PCR tube by mixing: Reagent 1 rxn (µl) 10 rxns + 1 extra Digested DNA sample 80 - Hybridization Solution ( ) Primer Cassette 1 11 HaloPlex Probes ( ) Total volume: Vortex the reaction briefly and spin down. 10

12 HALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL Step 2. Incubate over night 2.1 Incubate using a thermal cycler with the following program: C for 10 min C for 30 min C for 30 min C for 10 h C for 30 min 7. 8 C C for 30 min Practical Note: Thermal cyclers that use calculated temperature methods cannot be set to 160 μl reactions. In that case, enter the maximum possible volume. PHASE III - SOLID PHASE CAPTURE AND DNA LIGATION The HaloPlex probes contain biotin, and can thus be captured on streptavidin coated paramagnetic beads. Using a magnet rack, the targeted fragments are captured along with probes whereas other parts of the genome can be discarded by removing the supernatant. Washing the beads in step 2 serves to further enrich the captured fragments. Fragments correctly hybridized to their corresponding HaloPlex probe form perfect templates for a DNA ligase. Ligation results in the formation of closed DNA circles of targeted fragments. Note: Remove the Bead Solution and Wash Solution from -20 C storage and allow to reach room temperature before continuing. Step 1. Prepare the Magnetic Beads 1.1 Add 1 volume (20 µl per sample + extra) Magnetic Beads ( ) to a PCR tube and place it on the ring magnet rack for 5 min. 1.2 Discard the supernatant using a pipette. 1.3 Add 2 volumes of Capture Solution ( ). 1.4 Resuspend the beads by careful vortexing. 1.5 Use the washed beads immediately. 11

13 Version 1.0.3, April For research use only Step 2. Add the Magnetic Beads 2.1 Prepare the capture reaction by mixing: Reagent 1 rxn (µl) 10 rxns + 1 extra Prepared Magnetic Beads Hybridized DNA sample Total volume: Vortex briefly and incubate at room temperature for 15 min. Pipette 2.3 Spin the tubes for 5 seconds using a benchtop centrifuge. Reaction tube 2.4 Place the tubes in the magnet rack for 30 seconds. Magnet 2.5 Carefully remove and discard the supernatant using a pipette. 2.6 Proceed immediately to the next step. Step 3. Wash the beads 3.1 Remove the magnet rack and add 200 μl Wash Solution ( ) to each bead-containing tube. Vortex the tube thoroughly to resuspend the beads and incubate at 46 C for 30 min in a thermal cycler. Resuspend the beads by gentle vortexing every 10 min to avoid precipitation. Magnetic beads Practical note: to ensure that all supernatant is efficiently removed, set the pipette for 70 μl and insert the tip straight down until it hits the bottom of the tube. Slowly aspirate while keeping the tip in contact with the bottom of the tube. The extra volume setting on the pipette ensures that bubbles and leftover droplets of supernatant will be removed as well. The magnetic beads should remain safely located on the sides of the tube, however if some beads are removed along with the supernatant it is not a problem. 3.2 Spin the tubes for 5 seconds. 3.3 Place the tubes in the magnet rack for 30 seconds. 3.4 Carefully remove and discard the supernatant using a pipette. 3.5 Proceed immediately to the next step. 12

14 HALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL Step 4. Ligate the target fragments 4.1 Prepare a DNA ligation mix by combining: Reagent 1 rxn (µl) 10 rxns + 1 extra Ligation Solution ( ) DNA Ligase ( ) Total volume: Add 50 μl Ligation master mix to each reaction. 4.3 Vortex for 5 seconds to resuspend the beads. 4.4 Incubate at 55 C for 10 min in a thermal cycler. 4.5 Spin the tubes for 5 seconds. 4.6 Place the tubes in the magnet rack for 30 seconds. 4.7 Carefully remove and discard the supernatant using a pipette. 4.8 Proceed immediately to the next step. PHASE IV - ENRICHMENT BY PCR Step 1. Haloase A treatment 1.1 Prepare the Haloase A master mix by combining: Reagent 1 rxn (µl) 10 rxns + 1 extra Haloase A Buffer Haloase A 2 22 Total volume:

15 Version 1.0.3, April For research use only 1.2 Add 25 µl of the master mix to each sample, mix and incubate at 37 C for 30 min in a thermal cycler. 1.3 Spin the tubes for 5 s 1.4 Place the tubes in the magnet rack for 30 seconds. 1.5 Carefully remove and discard the supernatant using a pipette. 1.6 Proceed immediately to the next step. Step 2. Haloase B treatment 2.1 Add 21.5 µl of the Haloase B buffer to each sample. 2.2 Incubate at 80 C for 20 min in a thermal cycler to inactivate any residual Haloase A. 2.3 Remove the tubes from the thermal cycler and wait for them to cool down to room temperature. 2.4 Add 3.5 µl Haloase B to each sample (without removing the buffer), mix and incubate at 37 C for 30 min in a thermal cycler. 2.5 Spin the tubes for 5 s 2.6 Place the tubes in the magnet rack for 30 seconds.! 2.7 Carefully transfer the 25 µl supernatant to new tubes. 2.8 Keep the reactions on ice while preparing the PCR mixture. Please note that the DNA is not stable in the current conditions and should not be stored for any longer period of time. 14

16 HALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL Step 3. Amplify targeted fragments with PCR 3.1 Prepare the PCR reaction mix by combining: Reagent Stock konc. 1 rxn (µl) 10 rxns + 1 extra Collected sample 25 - Phusion HF Buffer 5 X 5 55 dntp 25 mm Illumina Primer µm 1 11 Illumina Primer µm 1 11 Phusion Polymerase 2 u/µl Purified H2O Total volume: Add 24 µl of the master mix to each sample. 3.3 For multiplexing sample preparations add 1 µl of the appropriate Illumina PCR Index Primer to each sample. 3.4 Run the following PCR protocol in a thermal cycler: 98 C 30 s 98 C 10 s 65 C 30 s 27 cycles 72 C 30 s 72 C 5 min 8 C The enrichment procedure is now complete. The resulting product is a pool of amplicons originating from the targeted region. Each amplicon is comprised of one target insert surrounded by the general motifs of the Illumina sequencing primers, index barcode and bridge PCR primers. The amplicons range from bp in size ( bp inserts and 125 bp sequencing motifs). Before loading the samples to a sequencer they need to be purified, validated and pooled. The final product contains the targeted fragments (blue) surrounded by the Illumina paired end sequencing primers (black), the sample barcode (orange) and the library bridge PCR primers (yellow). 15

17 Version 1.0.3, April For research use only PURIFY, VALIDATE AND POOL Validation of restriction digestion efficiency It is recommended to run the eight restriction digestions of the RE Control DNA on a gel to validate digestion. See Appendix for protocol and example data. Purify the enriched samples Remove any remaining primers by purifying the PCR products with Ampure XP beads. 1. Add 1.5x volumes of Ampure XP beads to each sample (e.g. 75 µl beads to 50 µl sample). 2. Pipette mix 10 times and incubate at room temperature for 5 min. 3. Place the tube on the magnet rack for 2 min and then discard the supernatant. 4. Keep the tube on the plate and add 200 µl freshly prepared 70% EtOH and incubate at room temperature for 1 min. 5. Discard the ethanol and repeat for a total of two washes. 6. Remove the tube from the magnet plate and add 40 µl Tris-Acetate buffer ph Pipette mix 10 times to elute the DNA. 8. Place the tube on the magnet rack for two minutes and then collect the supernatant. Validate library size It is recommended to run aliquots of the purified samples on a gel or an Agilent Bioanalyzer High Sensitivity DNA chip to verify correct size distribution. See figure 1 for example data. Figure 1. The size distribution of the enriched DNA samples is between bp. Assessment of enrichment using the QualityControl qpcr Kit To validate enrichment of the targeted region the enriched samples can be analyzed using a qpcr assay with primer pairs towards both enriched and non-enriched regions. The supplied QualityControl qpcr Kit contains seven on-target primer pairs and one off-target primer pair. Non-enriched genomic control DNA is also supplied as reference. See Appendix for protocol and example data. Pool samples with different barcodes If samples have been enriched with different barcodes they can be pooled to minimize the number of purification reactions in the next step. Pool equimolar amounts of each sample to optimize the use of sequencing capacity. 16

18 HALOPLEX PCR TARGET ENRICHMENT & LIBRARY PREPARATION PROTOCOL APPENDIX Validation of restriction digestion efficiency The RE Control DNA that was digested along with the ten DNA samples can be analyzed using gel electrophoresis to validate digestion efficiency. Each enzyme in the double digest reactions cut the RE Control DNA at a specific position creating a distinct pattern of differently sized fragments. Recommended protocol To generate the example data presented in figure 1 below, 3 µl from each of the eight restriction digestion reactions were run on a Novex Pre-Cast 6% PAGE gel (Invitrogen, EC62652BOX) at 210V for 15 min on the XCell SureLockTM Mini Cell electrophoresis system (Invitrogen). 40 ng of the undigested RE Control DNA sample and 300 ng 100 bp ladder (Invitrogen, ) were also run as references. The gel was post stained in 3x Gel- RedTM solution (Biotium) for 10 min before analysis under UV light. Any deviations from the patterns shown in figure 1 indicate either inefficient digestion or incorrect mixing of the double-digest enzymes. Assessment of enrichment using the QualityControl qpcr Kit The QualityControl qpcr Kit contains primer pairs that analyze either on- or off-target regions. Enrichment efficiency is validated by the cycle time threshold difference between on-target and off-target regions. Included reagents Seven on-target primer pairs (primers 1-7) One off-target primer pair (primer 8) Genomic Control DNA 100 ng/µl Additional required reagents and equipment SYBR Green PCR Master Mix ( , Applied Biosystems) (or other SYBR Green based reagents) ABI StepOne Plus Real Time PCR system (Applied Biosystems, or equivalent) Figure 1. Lane 1: 100 bp ladder, Lane 2: undigested RE Control DNA, Lane 3-10: RE Control DNA digestions 1-8. Recommended protocol Dilute the enriched DNA samples 1:1600 in 1x SYBR Green PCR Master Mix ( , Applied Biosystems) and 250 nm of respective primer pair to a total volume of 20 µl. For the Genomic Control DNA use 10 ng as template in the same PCR mixture as described above. To acquire the example data below the following cycling conditions were used on an ABI StepOnePlus Real Time PCR system: 95 C for 10 min followed by 40 cycles of [95 C 15s, 56 C 30s] with end-point measurement of the fluorescence after each completed cycle. 17

19 Version 1.0.3, April For research use only Analysis guidelines The ABI StepOnePlus Real Time PCR software was used to manually set a static threshold for all primers and samples at the middle of the exponential phase. The Cycle Time threshold values (Ct) were extracted and plotted in Figure 1. Table 1 below describes some key performance parameters that can be calculated from the data generated in this assay and how they should be interpreted. The average on-target Ct values can also be used when pooling indexed samples before sequencing. Ct value Enriched sample Genomic control DNA qpcr primer pair 1 qpcr primer pair 2 qpcr primer pair 3 qpcr primer pair 4 qpcr primer pair 5 qpcr primer pair 6 qpcr primer pair 7 qpcr primer pair 8 Figure 1. Plot of the Ct values from one enriched sample and from the Genomic DNA using seven on-target primer pairs (green) and one off-target primer pair (red). QC Parameter Guideline value Deviations Possible cause Solution qpcr standard deviation on the Genomic Control DNA < 1.5 Ct >1.5 Ct on the Genomic Control DNA Variable pipetting when setting up the qpcr Repeat the qpcr assay until successful qpcr standard deviation on any of the 10 enriched samples < 1.5 Ct >1.5 Ct on up to five of the enriched samples while gdna <1.5 Ct >1.5 Ct on more than five of the enriched samples while gdna <1.5 Ct Variable pipetting when setting up the qpcr Inefficient amplification - carefully remove all supernatant prior to amplification Repeat the qpcr assay. If problem persists repeat enrichment of failed samples Repeat the qpcr assay. If problem persists repeat enrichment of failed samples Off-target primer pair Ct on enriched samples >30 <30 on up to five of the enriched samples <30 on more than five of the enriched samples Contamination while setting up the qpcr assay Contamination of genomic DNA during the enrichment Repeat the qpcr assay. Repeat the qpcr assay. If problem persists repeat enrichment of failed samples 18

20 CONTACT Halo Genomics AB Dag Hammarskjölds Väg 54A SE Uppsala SWEDEN Tel Fax Copyright 2011 Halo Genomics AB. All rights reserved. Conditions of use: This product is for research use only. Not for use in diagnostic procedures. Trademarks: HaloPlex is a trademark of Halo Genomics. All other trademarks are trademarks of their respective owners. HaloPlex PCR Technology is protected by several patents and patent applications. Restriction and Liabilities: This document is provided as is. Halo Genomics assumes no responsibility for inaccuracies or typographic or technical errors in the document.