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					version 2005-03-22


attomol® lactose intolerance -13910C>T
Quicktype
Mutation assay for the detection of the transition -13910C>T
upstream of the human lactase-gene
For in vitro diagnostic use only!
20 determinations               order number: 1045

1. Introduction
The uptake of milk and other lactose-containing foods causes indigestibilities in some adults. Such symptoms are
often assigned to the syndrome of the hereditary lactose intolerance (LIT) that manifests in adults. The most fre-
quent 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 an activating mutation in the MCM6 gene (minichromosome maintenance
gene) on chromosome 2, near the lactase gene (lactase phlorizin hydrolase gene, LPH gene). The mutation, that
can be detected using this assay, is a base exchange from C to T at the nucleotide position -13910 upstream of
the lactase gene. Homozygous carriers of the mutation have a livelong lactose tolerance. In heterozygous carriers
the lactose intolerance may be partially compensated (Obermayer-Pietsch, 2004, Journal für Mineralstoffwechsel,
11(3):20-23; Sibley, 2004, Am J Pharmacogenomics, 4(4):239-245).
Wild type carriers develop a lactase deficiency after breastfeed that proceeds with age leading to lactose intoler-
ance. As a consequence of lactase deficiency, lactose is not effectively cleaved yielding the monosaccharides
glucose and galactose that causes the above mentioned symptoms (Srinivasan und Minocha, 1998, Postgraduate
Medicine, 104(3):109-111, 115-116, 122-123). Only the monosaccharides can be reabsorbed during digestion.
The prevalence of the lactose intolerance varies world-wide strongly between different ethical groups. In the
European population, the lactose intolerance is with 30 % not so widely distributed (Sahi, 1994, Scand J Gastro-
enterol, 202(29):7-20). In Austria for example, 20-25 % of the people suffer from the primary adult lactose in-
tolerance (Obermayer-Pietsch, 2004, Journal für Mineralstoffwechsel, 11(3):20-23).

2. General Remarks
    •     This kit should only be used for in vitro diagnostic applications.
    •     Do not use this kit after the expiration date.
    •     To carry out the assay take care for an appropriate laboratory equipment and well-trained staff.
    •     Patients samples should be concerned as a potentially infectious agent and should therefore be handled
          according to the current law.
    •     Store reagents for the PCR* apart from patient DNA samples and amplification products.

3. Materials included
        Order     Reagent                                                  Volumes                                 Storage
        Number                                                                                                   Temperature
           38     deionised PCR-H2O                                      0.2 ml                                         C
                                                                                                                    -20 °
           83     PCR-buffer I                                           0.5 ml                                         C
                                                                                                                    -20 °
                  Primer LIT -13910C>T              2 tubes containing lyophilised primers, dissolve
         156                                                                                                                C
                                                                                                                        -20 °
                  (violet coloured tubes)               with 225 µl PCR-buffer I (#83) per tube
    The expiration date of all reagents included in the kit, is on the main label attached to the outside of the box.

4. Additional Reagents and Materials not included
    •     Patients DNA sample (use genomic DNA, concentration app. 20 ng/µl, dissolved in aqua dest. or TE-
          buffer)
    •     HotStarTaq DNA Polymerase provided by Qiagen
    •     Control template (-13910C>T heterozygous DNA)
    •     Eppendorf-tubes (0.2 ml; 1.5 ml)
    •     Mikropipettes (volume range 0.5-1000 µl) and sterile filter tips
    •     Thermocycler
    •     Equipment for agarose gel electrophoresis and consumables (agarose or precasted 3 % agarose gels,
          electrode buffer, sample buffer, DNA-dye, UV-transilluminator)

5. Test Principle
During the quicktype genotyping process, both alleles of a certain gene become amplified specifically. The
examination of the amplification products can be performed by agarose gel electrophoresis.
If both alleles carry the wild type sequence (homozygous wild type) the agarose gel contains only one specific
band at 290 bp. If one allel carries the wild type sequence and the other the mutation sequence (heterozygous)
there will be two bands at 290 bp and 330 bp, respectively. If the sample DNA is homozygously mutated, only one
band appears at 330 bp in the gel. For calibrating the bands in a gel you should use a heterozygous sample DNA
as a reference for a better justification of the results (see fig. 1).




*) PCR is a patented procedure hold by Hoffmann-La Roche. The purchase of this kit does not include a licence for PCR.
version 2005-03-22



6. Manual
It is recommended to include the following set of control PCR reactions in addition to patients samples:
1. Negative control (PCR-blank)
2. Positive control (use a heterozygous control template [genotyped, genomic DNA])
The control template may be ordered separately from the manufacturer (order number 160). Patients samples
that has been genotyped correctly by the customer can also be used as control template.

Protocol:
•    DNA-preparation from patients blood according to standard procedures

•    Solubilisation of the primer with 225 µl PCR-buffer I (order number 83) per tube, thoroughly mixing by
     aspirating and dispensing using a pipette, and incubation at room temperature for 5 min results in the PCR
     premix
                                                                                                         C
     Attention: If you need less than 225 µl PCR premix, you can store the rest of the PCR premix at -20 ° .

•    Preparation of the PCR mix by addition of the hot start Taq DNA polymerase to the PCR premix:
     The amount of the PCR premix used for one reaction (20 µl) has to be multiplied with the number of samples
     and controls in the series plus a sufficient excess volume of premix (example for 8 reactions: 8 x 20 µl + 10 µl
     excess volume = 170 µl PCR premix).
     Thereafter, the hot start Taq DNA polymerase (5 U/µl) is added to the premix in a final dilution of 1:100 (for
     example: 170 µl PCR premix + 1.7 µl 5 U/µl polymerase). Now, the PCR mix has to be mixed thoroughly.

•    Aliquot the PCR mix into 0.2 ml-tubes:
              Negative control:                       Positive control:                         Patients sample:
               20 µl PCR mix                            20 µl PCR mix                            20 µl PCR mix
                                                   + 5 µl control template
                + 5 µl PCR-H2O                                                            + 5 µl DNA (ca. 20 ng/µl)
                                                        (ca. 20 ng/µl)

•    PCR is performed in a thermocycler using the temperature regime listed below:
             initial denaturation:  15 min 95 °C
                          5 cycles: 1 min 94 °C          1 min 63 °C        1 min 72 °C
                         30 cycles:         C
                                    30 s 94 °                    C
                                                         30 s 63 °                  C
                                                                            30 s 72 °
                   final synthesis: 2 min 72 °C
                              hold:   C
                                    4 ° without time limit

•    Analysis of the PCR products is performed by transferring 10 µl of each PCR reaction onto an agarose gel
     (2.5 - 3 %) and subsequent electrophoresis according to laboratory standards

•    Genotyping of the patients samples is done by comparing the bands pattern of the samples with that of the
     controls (see fig. 1)
                                                                 Negative   Positive   sample    sample   sample
                                                                  control   control      1         2        3
     Genotypes:                                                               HZ        MU        WT        HZ
     WT = homozygous Wild Type = Lactose intolerant
     HZ = HeteroZygous
     MU = homozygous MUtation = Lactose tolerant




                                                                 Fig. 1.: Typical picture of quicktype-amplified samples
                                                                          DNA separated in a 2.5 % agarose gel


In the case of problems during the test application do not hesitate to contact the manufacturer or the distributor.


attomol GmbH                                     Tel.: +49 35329 56081
Molekulare Diagnostika                           Fax: +49 35329 56080
Schulweg 6                                       e-mail: info@attomol.de
03205 Lipten
Germany

				
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posted:12/22/2011
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