Recently, three ample -to-be trials absolutely approved that the appraisal of basal balance ache (MRD) plays a cardinal role as a anxiety constant in astute lymphoblastic leukemia (ALL).123 Individualized ameliorative adaptations based on the MRD cachet ability be of ample account for ALL patients. Several methodologies are accessible for MRD analyses in ALL patients.1234 Immunoglobulin (Ig) and T corpuscle receptor (TCR) gene rearrangements accept been activated as PCR targets. An important PCR action for MRD assay is the accession of the rearranged Ig or TCR genes and admixture with 32P-labeled clone-specific probes in dot blemish hybridization. Now that the appliance of MRD ecology in ALL has been recognized, modification of accepted techniques or development of new techniques enabling accelerated and reliable MRD assay is relevant. We address on a non-radioactive dot blemish assay demography advantage of digoxigenin (DIG)-labeled oligonucleotides for the accelerated apprehension of MRD. DIG is a steroid hapten which is affiliated via a spacer arm to a nucleotide. DIG-ddUTP is congenital at the 3′-end of an oligonucleotide by terminal transferase. Afterwards hybridization, the ambition DNA is apparent by an enzyme-linked immunoassay through an anti-DIG-AP Fab fragment.
We activated this address on 10 developed ALL patients with IGK and/or TCRD gene rearrangements. The patients were advised according to the German multicenter balloon of ALL in adults 05/99.5 High atomic weight DNA was able from cartilage bottom samples acquired at antecedent analysis application accepted techniques. IGK and TCRD gene rearrangements were articular and characterized by PCR and sequencing as described.67 Oligonucleotides agnate to the clone-specific junctional regions of the corresponding IGK/TCRD genes are apparent in Table 1.
Ten-fold consecutive dilutions of antecedent leukemic DNA into accustomed buffy covering (BC) DNA were able in a ambit from 10−1 to 10−6. One μg of anniversary DNA alertness as able-bodied as BC DNA were amplified application a set of alien primers.6 A abrogating ascendancy (H2O) was included in anniversary PCR reaction. PCR was performed application the blow bottomward affairs with 40 cycles.7 Bristles μl of the articles were spotted in alike on to nylon blur (NYTRAN 0.45, Schleicher and Schuell, Dassel, Germany). The membranes were blood-soaked with denaturing absorber (0.5 M NaOH, 1.5 M NaCl) and acrid absorber (0.5 M Tris-HCl, 1.5 M NaCl, pH 7.0) for 2 min anniversary and cross-linked by UV.
100 pmol of anniversary oligonucleotide was 3′-end labeled with DIG-ddUTP application a DIG oligonucleotide 3′-end labeling kit (Roche Atomic Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. Non-incorporated DIG-ddUTPs were removed by booze precipitation and the labeled delving was attenuated into 20 μl of distilled baptize (final absorption 5 pmol/μl). One pmol of the DIG-labeled oligonucleotide was spotted on to a nylon membrane, which was afterwards candy in the immunological apprehension footfall to verify the labeling efficiency. The membranes were prehybridized at 50°C for 30 min in a artificial box absolute 20 ml admixture solution, ie 5 × SSC, 1.0% (w/v) blocking reagent (Roche Atomic Biochemicals), 0.1% N-lauroylsarcosine, 0.02% SDS. The membranes were again alloyed in a closed artificial bag with 3 ml admixture band-aid and 6 μl of the labeling delving (30 pmol), demography affliction that no air balloon was present. The membranes were incubated for 90 min at the optimal temperature depending on the melting temperature (Tm) of the corresponding oligonucleotide. The membranes were done alert in 2 × SSC, 0.1% (w/v) SDS at allowance temperature for 5 min, followed by an added ablution in a 0.1–1 × SSC, 0.1% (w/v) SDS band-aid at the admixture temperature. The immunological apprehension was performed as follows. The membranes were blood-soaked with blocking band-aid (0.1 M maleic acid, 0.15 M NaCl, 1% blocking reagent, pH 7.5) for 30 min at allowance temperature. Several membranes can be candy accompanying in the aforementioned artificial box. The band-aid was alone and 30 ml of blocking band-aid absolute 6 μl of anti-DIG-alkaline phosphatase Fab bits (Roche Atomic Biochemicals) was added, followed by an added evolution for 30 min. Finally, the membranes were done alert with abrasion absorber (0.1 M maleic acid, 0.15 M NaCl, 0.3% Tween 20, pH 7.5) for 15 min and incubated with apprehension absorber (0.1 M Tris-HCl, 0.1 M NaCl, 50 mM MgCl2, pH 9.5) for 2 min. Anniversary blur was placed on a artificial admixture bag and bristles drops of chemiluminescence substrate (CSPD ready-to-use, Roche Atomic Biochemicals) were added on to the apparent of the membrane. The admixture accoutrements were closed and incubated at 37°C for 10 min. Autography was performed application an X-OMAT DS blur (Kodak) for 2–10 min. For the radioactive dot blemish hybrization method, 20 pmol of anniversary oligonucleotide was end-labeled with γ-32P-ATP application a T4 polynucleotide kinase (Amersham Pharmacia Biotech, Freiburg, Germany) at 37°C for 45 min. Admixture and abrasion altitude were ahead described.6
The sensitivities for corresponding PCR targets application both methods are abbreviated in Table 1 and adumbrative after-effects are apparent in Figure 1. It can be assured that the acuteness of the DIG-based adjustment was commensurable to that of the radioactive dot blemish admixture method. Both methods acquiesce the assurance of the absorption of an alien cartilage bottom aftereffect sample.
Dot blemish hybridizations of accommodating No. 36. The acuteness of the DIG-labeled Vδ2-Dδ3 oligonucleotide akin that of the 32P-labeled oligonucleotide. With account to the VκII-Kde rearrangement, the apprehension absolute of the DIG-based adjustment accomplished 10−5, admitting the radioactive adjustment showed a acuteness of 10−4.
There are another procedures for DIG-labeling of oligonucleotides such as 5′-labeling or 3′-tailing. However, the aboriginal adjustment requires the accession of an aminolinker at the 5′ end of the oligonucleotide at the final footfall of the oligonucleotide synthesis. The additional adjustment adds a appendage of 50 bp to the oligonucleotide which ability abate its specificity. Recently, a new labeling kit based on the absolute labeling of oligonucleotides with AP via a 5′-aminolinker has become available, which makes the immunological footfall superfluous. Although the closing allows a added accelerated apprehension of alloyed oligonucleotides, we absitively to stick with the DIG oligonuclotide 3′-end labeling kit for economical affidavit alone (kit prices).
Compared to the radioactive method, the DIG-based adjustment bears several advantages. Firstly, the action is non-radioactive and can accordingly be performed in any accepted laboratory. Secondly, due to the abbreviate acknowledgment time (2–10 min) compared to that of the radioactive delving (12 h to several days), the accomplished action can be beneath considerably. Thirdly, end labeling of the oligonucleotide can be done at any time and the corresponding probes can be stored at −20°C until use. We commonly blend at an optimal absorption of 10 pmol/ml, implying that the labeled delving can be acclimated up to four times because a clarify with a admeasurement of 25 cm2. Fourthly, the admixture band-aid of DIG-labeled probes could be re-used several times, provided that the band-aid has been stored at −20°C. Hence, this adjustment can save money as able-bodied as time. A accessory disadvantage of the DIG-based adjustment is that bargain arresting acuteness is sometimes empiric due to overwashing afterwards hybridization. Since it is accessible to ablution the filters again for optimal results, the aboriginal abrasion footfall should be performed beneath low acerbity condition.
Recently, a absolutely automatic arrangement for the sequence-specific and semiquantitative apprehension of PCR articles has been published.8 The key to the arrangement is the use of target-specific microbeads, accumulation covalently absorbed oligonucleotide probes. PCR samples are automatically analyzed application the absolute aqueous handling, temperature ascendancy and arresting apprehension capabilities of the system. A abundant advantage of this arrangement is that hybridization, abrasion and apprehension procedures are automated. Thus, arduous abstracts for admixture can be omitted. However, its acuteness and account for the appraisal of MRD in ALL has not been advised yet.
Recent abstracts acquired from a ample alternation of adolescence ALL acerb advance that the time-points 1 and 3 months afterwards consecration analysis aggregate analytical ambit for the appraisal of ameliorative effectiveness.3 The aing claiming will be to analysis whether MRD-oriented ameliorative modifications can absolutely advance the aftereffect of ALL. Demography this assignment into consideration, a accelerated and reproducible adjustment is required. We anticipate the DIG adjustment fulfills these criteria.
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