lactose intolerance C>T-Realtime LT 2 - version

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1 lactose intolerance C>T-Realtime LT 2 - version attomol lactose intolerance C>T- Realtime LT 2 Assay for the detection of the transition C>T upstream of the human lactase gene using LightCycler with differentiation from possible neighboring polymorphisms For in vitro diagnostic use only! 40 determinations Order number: 1188 C 1. Introduction The uptake of milk and other lactose-containing food may causes indigestibilities. Such symptoms are often assigned to the syndrome of the hereditary lactose intolerance (LIT) that manifests in adults. The most frequent symptoms are lactose maldigestion, meteorism (tympanites), distension, and diarrhoea (Obermayer-Pietsch, 2004, Journal für Mineralstoffwechsel, 11(3):20-23). Lactose tolerance may be caused by activating mutations in the MCM6 gene (minichromosome maintenance gene) on chromosome 2, near the lactase gene (lactase phlorizin hydrolase gene, LPH gene) (Olds und Sibley, 2003, Hum Mol Genet, 12(18): ). Activating mutation denotes a mutation where the carrier receives a biological advantage compared to the non-mutated individual. In this example, it is the tolerance towards lactose. The mutation, detectable through this assay, is a base exchange from C (wild type) to T (mutation) at the nucleotide position upstream of the lactase gene. Homozygous carriers of the mutation have a livelong lactose tolerance. In heterozygous carriers it is supposed that the lactose intolerance may be partially compensated (Obermayer-Pietsch, 2004, Journal für Mineralstoffwechsel, 11(3):20-23; Sibley, 2004, Am J Pharmacogenomics, 4(4): ). Wild type carriers develop a lactase deficiency after breastfeed that proceeds with age leading to lactose intolerance. As a consequence of lactase deficiency lactose is not cleaved effectively into the monosaccharides glucose and galactose. Because not-cleaved lactose can not be reabsorbed by the human organism the symptoms mentioned above can typically be found (Srinivasan und Minocha, 1998, Postgraduate Medicine, 104(3): , , ). The loss of lactase activity usually starts after the ablactating and ends in adult at the status of lactose intolerance. The prevalence of the lactose intolerance varies world-wide strongly between different ethnical groups. In the European population, with a prevalence of less than 30 %, the lactose intolerance is not so widely distributed (Sahi, 1994, Scand J Gastroenterol, 202(29):7-20). In Austria for example, % of the people suffer from the primary adult lactose intolerance (Obermayer- Pietsch, 2004, Journal für Mineralstoffwechsel, 11(3):20-23). There are other possible mutations known in the direct neighborhood of position , for example the mutations C/G, T/C, G/A, and T/G. These mutations may occur more often at individuals of African or Arabic origin (Tag et al, 2007, Clin Chem, 53(1): ; Torniainen et. al, 2009, BMC Genet, 10(31)). The importance for lactose tolerance differs depending on the specific mutation. 2. Important Remarks Keep the kit at the indicated storage temperature. This kit should be used for in vitro diagnostic applications only. Always use the added manual for test application. Do not use this kit after the expiration date. Ensure that only well-trained staffs in an appropriately equipped laboratory carry out the assay. Wear gloves during all steps of the test application. Patient s samples should be considered as a potentially infectious agent and should therefore be handled according to the current law. Store reagents for the PCR well apart from patient DNA samples and amplification products.

2 lactose intolerance C>T-Realtime LT 2 - version Materials included Order number 314 Reagent Volumes/quantity Storage temperature Oligomix LIT C>T LT 2 (red cap) 2 tubes with 40 µl primer-/probe mixture each, ready to use -20 C until first use, after first use store tubes dark at 4 C* * Freezing of oligomix after first use is recommended only if you do not plan to use it during the next 2 weeks. 4. Additional Reagents and Materials not included Patient s DNA samples (use genomic DNA dissolved in aqua dest. or TE-buffer) Control template (LIT C>T heterozygous DNA) Micropipettes (volume range µl) and sterile filter tips parts of the kit LightCycler Faststart-DNA-Master HybProbe (Roche, order no or ): mix vial no. 1a (red cap) with vial no. 1b (colorless cap) according to instructions of Roche, resulting in 10x ready to use mix (contains Taq polymerase, nucleotides, MgCl 2 ) hereinafter called Roche Mix LightCycler -capillaries (20 µl, for LightCycler 1 or 2; order no ) or suitable PCR tubes for LightCycler 480 LightCycler 1.x, 2.0 or 480 with appropriate control and evaluation software Centrifuge for capillaries and PCR tubes respectively 5. Test principle After isolating DNA from patient s samples, both alleles of a certain gene become amplified within one reaction mixture. Detection of amplification products takes place by hybridization of the specific mutation-covering LoopTag* ) probe. The LoopTag probe hybridizes to the elongated primer forming a primer-probe loop. The formation of a loop enables FRET between the fluorescence donor (D) and the fluorescence acceptor (A) as shown in fig. 1. Dehybridization of LoopTag probe during melting curve analysis reveals melting peaks at different temperatures indicating wild type and mutated template sequences. If both alleles at position carry the wild type sequence (homozygous wild type) a melting curve with 1 peak results at 53.0 ± 2 C. If one allele carries the wild type sequence and the other the mutation sequence (heterozygous) there will be a melting curve with 2 peaks (53.0 and 57.0 ± 2 C). If the sample DNA is mutated homozygously, only one peak at 57.0 ± 2 C can be found. If there exists a mutation at one of the positions , , or a melting peak occurs at 46.5 ± 2 C. These additional mutations can be clearly separated from the mutation at position but can not be distinguished in between (see explanation to figure 2). To differentiate the additional mutations from each other a second test procedure has to be included (e.g. sequencing). A clinical diagnosis never should be concluded from results of one diagnostic method alone. The doctor has to consider all clinical and laboratory results available. 3 5 A primerelongation A FRET D 3 Fig. 1: LoopTag detection principle probe hybridization * ) LoopTag is patented by Attomol (patent-no. PCT/EP2008/057512).

3 lactose intolerance C>T-Realtime LT 2 - version Manual It is recommended to include appropriate controls in each sample series: 1. Negative control (PCR blank) 2. Positive control (heterozygous for ) The control template may be ordered separately from the manufacturer (order no. 160). Patient s samples that were genotyped correctly by the customer can also be used as control template. Protocol: DNA preparation from patient s blood according to standard procedures; resulting DNA samples (app. 20 ng/µl) can be used without dilution. Attention: If you have any problems (poor amplification, not evaluable melting curves) at first repeat the assay with diluted DNA samples (e.g. 1:10). Thaw tubes with oligomix, shake and centrifuge the tubes to pull down condensed water. Storage remark: After taking off the needed amount of oligomix you can store the rest of it in the refrigerator (4 C, dark). Freezing of oligomix is recommended only if you do not plan to use it during the next 2 weeks. Before you take it again please centrifuge and mix it well. Generally avoid repeated freezing-thawing-cycles of the oligomix! Preparation of the PCR mix (8 µl PCR mix und 2 µl sample DNA are required for one PCR reaction): According to the number of samples pipette, Roche mix, and oligomix in an Eppendorf tube for the preparation of PCR mix (see following table). Mix it well after preparation. number of samples Roche mix oligomix number of samples Roche mix oligomix Transfer of reagents to LightCycler capillaries or tubes Negative control: Positive control: Patient s sample: 8 µl PCR mix 8 µl PCR mix 8 µl PCR mix + 2 µl + 2 µl control template + 2 µl DNA Seal the capillaries/tubes and centrifuge. PCR incubation of the samples is performed in the LightCycler using the programs listed in Appendix 1. Evaluation of the samples is done by analysis of number and position of peak maxima as described in figure 2.

4 lactose intolerance C>T-Realtime LT 2 - version Fig. 2: Example for the evaluation of melting curves of - a homozygous wild type sample (-13910C/C = lactose intolerance, blue curve) - a heterozygote sample (-13910C/T, green curve) - a homozygote mutated sample (-13910T/T = lactose tolerance, red curve) - a sample with a heterozygote constellation T/G and a homozygote wild type C/C (ocher curve) - a sample with a heterozygote constellation G/A and a heterozygote constellation C/T (brown curve) - negative control (black curve) Assistance to figure 2 to evaluate the received melting curves allele 1 allele 2 Melting curves peak(s) at other mut.* other mut.* (tolerance level each ± 2 C) Curve color WT (C) WT WT (C) WT 53 C blue MU (T) WT WT (C) WT 53 C 57 C green MU (T) WT MU (T) WT 57 C red WT (C) MU WT (C) WT 46.5 C 53 C ocher WT (C) MU MU (T) WT 46.5 C 57 C brown WT (C) MU WT (C) MU 46.5 C not shown * other mut.: means a mutation at position , , or Depending on the applied LigthCycler model and the circumstances among the device runs during the melting curve analysis it can sporadically happen that the melting temperatures of the peaks lie outside of the tolerance region. Therefore, it is recommended to include a heterozygous control as a reference in each sample series. In the case of problems during test application do not hesitate to contact the manufacturer or the distributor.

5 lactose intolerance C>T-Realtime LT 2 - version Appendix 1 The wavelengths necessary for excitation and detection can be found in the following table: LC 1.x LC 2.0 LC 480 excitation automatic automatic 483 or 498 nm detection 640/510 nm 640/530 nm 640 nm To perform the PCR and melting curve analysis use the LightCycler programs in the following tables. Program for LC 1.x and 2.0 Program for LC 480 Program: Activation; Type: None; Cycles: 1 Program: Activation; Analysis : None; Cycles: 1 Temperature Hold Time Slope Target Hold Ramp Rate Target ( C) (sec) ( C/sec) ( C) (s) ( C/s) none 95 none s Program: Amplification; Type: Quantification; Cycles: 40 Program: Amplification; Analysis : Quantification; Cycles: 40 Temperature Target ( C) Hold Time (sec) Slope ( C/sec) Target ( C) Hold (s) Ramp Rate ( C/s) none 95 none single 55 single none 72 none s Program: Melting curve; Type: Melt. Curves; Cycles: 1 Program: Melting curve; Analysis : Melt. Curves; Cycles: 1 Temperature Target ( C) Hold Time (sec) Slope ( C/sec) Target ( C) Hold (s) Ramp Rate ( C/s) none 95 none none 50 none none 40 none step 80 continuous s Program: Cooling; Type: None; Cycles: 1 Program: Cooling; Analysis : None; Cycles: 1 Temperature Hold Time Slope Target Hold Ramp Rate Target ( C) (sec) ( C/sec) ( C) (s) ( C/s) none 40 none s Appendix 2 Overview about symbols used for labeling of tubes and kit. explanation of symbols h manufacturer l batch number v expiry date 2 t 6 store between +2 C and +6 C o -20 store at a maximum of -20 C b catalogue number i in vitro diagnostic medical devices pn content sufficient for <n> determinations g consult instructions for use