DE3935022A1 - POLYNUCLEOTID PROBE - Google Patents

Abstract

A method for the assessment of sample DNA fragmentation which comprises detecting the presence or absence of a control fragment formed, during sample DNA cleavage, after one or more of the DNA fragments to be characterised. Polynucleotides and polynucleotide probes for use in the method as well as control kits comprising these. In particular the method of the invention is useful as a digestion control in methods of genetic characterisation. Probes binding to sequence 5'-ACATGGCAGG (AGGGCAGG)n TGGAGGG-3' where n= 1 or 2 are claimed.

Description

The invention relates to a method for determination the sample DNA fragmentation as well as on polynucleotides and Polynucleotide probes for use in this method. The The method according to the invention is particularly suitable for Digestion control in genetic characterization procedures.

Analytical methods have been developed to measure size and / or to determine the composition of DNA fragments. In particular, methods have been developed in which the DNA Fragments for hybridization to probes for one or several genetic sites are specific, made single-stranded will. The probes are typically used for identification purposes marked. The probes / fragment Hybrids are identified and characterized, for example in size using known techniques be such. B. Gel electrophoresis. Autoradiography is a Appropriate method for the identification of radiolabeled Probes / fragment hybrids.

The sample DNA fragmentation is different Process achieved such. B. Public address and chemical Activities. However, the sample DNA is usually cut enzymatically, for example by restriction enzymes or endonucleases. Restriction endonucleases are included capable of recognizing target nucleotide sequences in DNA the DNA in a predetermined manner with respect to the target sequence to cut. Such endonucleases are therefore considered analytical Tools usable. The enzymatic fragmentation of DNA is often referred to as "digestion".

The human genome has already been studied to be genetic Identify places that are informative. Polymorphs or hypervariable places have aroused special interest.  

The total number of such places is unknown, but it is probably big. In fact, the human genome contain at least 1500 such places. It has been shown that certain of these places in genetic engineering procedures Characterization are useful. In GB-PS 21 66 445 (Lister Institute of Preventive Medicine) are probes described the genomic DNA with respect to more than one can characterize informative genetic location. Probes, the genomic DNA for a single informational location characterize, were also developed and are described in EU-OS 2 38 329.

A problem that is often linked to analytical methods which are listed above and in which DNA fragments is that the extent of the sample DNA Fragmentation is not easy without complete knowledge of all relevant parameters can be determined or estimated. This includes the strength of the fragmentation measures, such as e.g. B. the enzyme concentration, the DNA sample concentration, the Buffer concentration and the temperature. It is therefore often necessary to let a long time pass before one complete or sufficient DNA fragmentation assumed can be. There are certain delays Inefficiency results, especially if many analytical tests are carried out. Beyond that it is desires that all informative DNA fragments, e.g. B. all DNA fragments resulting from complete digestion of the Sample DNA with one or more restriction enzymes result from the characterization, so that the maximum information obtainable from the fragments, such as. B. whose size and / or composition can be obtained.

The present invention is based on the finding that certain informative places in genomic DNA, for example are useful in genetic characterization methods, too are associated with areas of DNA that are fragmented resist. The identification of fragments, such Locations can therefore be used to create a DNA  To achieve fragmentation control in analytical processes.

The invention is therefore in one respect Method for determining sample DNA fragmentation, at which determines the presence of a control fragment that is during sample DNA cutting after formation one or more of the DNA fragments to be characterized is formed.

Sample DNA fragmentation is accomplished by any convenient Cutting processes such as B. public address or chemical Measures carried out, but it is preferably carried out under Use of one or more enzymes. For this reason the terms "sample DNA fragmentation" and "sample DNA cutting "used synchronously. Examples of suitable Are enzymes for use according to the invention Restriction enzymes such as e.g. B. Restriction endonucleases. The The present procedure has proven particularly useful as a digestive system Control proved to be useful if the restriction enzyme Hinf I is used. There is complete DNA fragmentation not essential for the implementation of the invention. It is only that the DNA control fragment according to the to be characterized Fragments is formed. The control fragment can therefore be selected to any desired Determine degree of fragmentation, such as. B. up to 50%, up to 60%, up to 70%, up to 80% or up to 90% of the fragmentation. However, the control fragment becomes useful this way selected that the control fragment is a complete sample Indicates DNA fragmentation. The sample DNA is useful Genomic DNA, such as B. Human DNA.

A control fragment can be of any convenient size or Composition, provided that it is within a fragmentation-resistant location is present. It will be on it noted that the expression "fragmentation resistant place" refers to a place that is better at fragmentation resists as one or more places that the to be characterized  Contain DNA fragments. A control fragment can be identified, for example, by DNA cleaves, for example using restriction enzymes, and that one follows the formation of DNA fragments. Fragments that contain a fragmentation-resistant location, disappear later in the fragmentation process, whereby one or more appropriate control fragments are formed will. The occurrence of a control fragment can be caused by any Appropriate measure for the determination of control fragments be determined. Once a control fragment is identified then it is not necessary to use the DNA Follow the fragmentation process continuously. In a more appropriate way A sufficient degree of fragmentation is estimated based on empirical observations, whereupon the control fragment is used to generate the Confirm the accuracy of the estimate.

It is believed that the inventive method in is useful in any situation in which the extent of Sample DNA fragmentation requires a determination, for example if sample DNA fragments by size and / or composition can be characterized. The procedure is particularly useful in genetic engineering procedures Characterization.

It has already been demonstrated that mini satellite areas or hypervariable locations in genomic DNA in genetic engineering procedures Characterization can be used. In the GB-PS 21 66 445 (Lister Institute of Preventive Medicine) Methods and probes for characterizing genomic DNA with respect to one or more mini satellite areas or described hypervariable places. In addition, the EU-OS 2 38 329 Methods and probes for the characterization of Genomic DNA in terms of individual mini-satellite regions or hypervariable places.

The method according to the invention is particularly useful if Sample genome DNA using mini-satellite region probes  or hypervariable locations is characterized. The Creation of a fragmentation control even allows one even greater level of security when interpreting results be such. B. in paternity and court cases.

Useful mini satellite probes are e.g. B. the multilocus probes 33.6 and 33.15, which are described in GB-PS 21 66 445 (Lister Institute of Preventive Medicine) and the single locus probes p Lambda g3, MS 1, MS 8, MS 31, MS 32 and MS 43, which in EU-OS 2 38 329 are claimed.

The determination of a control fragment is more expedient Way using polynucleotides or polynucleotide probes, those for hybridization to the control fragment are able. If the control fragment is a mini satellite area or contains a hypervariable place, then the polynucleotide or the polynucleotide probe hybridizes Appropriately to the area or location mentioned.

The control fragment is preferably probed by Sample DNA fragments with a mini satellite probe identified, with a single mini satellite region or hypervariable location is determined. The expressions "Mini satellite area" and "hypervariable location" used synonymously. The mini satellite probe can be used as Control probe can be viewed. This allows the Formation of a control fragment can be tracked more easily, because, for example, hybridization filters are easy to use Control probe to be probed.

Polynucleotides of DNA, RNA and any other kind of DNA are hybridizable, can be used. The polynucleotides are unmarked and can be in double strand form (ds) or in single strand form (ss). Generally speaking at least the nucleotide sequence of the polynucleotide in single strand form present, but preferably the probe will Polynucleotide completely contained in single strand form. Polynucleotides for use in the method according to the invention  are made useful by microbiological reproduction of cloned material or produced by direct synthesis.

Polynucleotide probes can be labeled or labeled obtained from polynucleotides. Labeled polynucleotides in ss-form can be used as probes what also applies to their marked predecessors, from which ss- Probes can be made. The according to the invention polynucleotide probes used are preferably radiolabeled, for example with ³²P or with ³⁵S, namely for example in any convenient way. The probes can alternatively characterized by non-radioactive measures be well known in hybridization technology are, such as B. with fluorescent groups or with biotin or derivatives thereof.

It should be noted that when polynucleotides or Polynucleotide probes used for hybridization to polymorphic or hypervariable places are capable of being used and if Restriction enzymes are used to cut the sample DNA then it is desired that the selected one Probe / enzyme combination is such that the enzymatic Cutting the DNA did not use the probe / fragment hybridization disturbs.

Digestion resistance was found in the neighborhood of the observed with MS 51 mini satellite area. This primarily affects cutting with Hinf I- Restriction endonuclease in the neighborhood of this mini satellite. Without being determined by theoretical considerations wanting, it is believed that the fragmentation resistance, for example the digestive resistance Changes in the tertiary structure of DNA can result. These alternative structures can, for example, by a special frequency of nucleotides, due to environmental conditions (pH, ion concentration) or long-term effects will. The properties of such alternative structures  were described by R. D. Wells and S. C. Harvey in "Unusual DNA Structures "(1988, Springer-Verlag). Changes The tertiary structure can have the assets of, for example a restriction enzyme, a nucleic acid binding site recognize, or its ability to cleave nucleic acid, influence. Nucleotide sequences of interest are those which, for example, in intra rank hybridization and especially play a role with non-Watson-Crick couples. Such nucleotide sequences can be G-rich, for example. For example, the term "G-rich" can refer to nucleotide sequences receive at least 50%, at least 60%, at least 70%, at least 80% or at least 90% Guanine residues included. Such changes in the tertiary Structure are useful in the paper by V. I. Lyamichev et al. in Nature, 339 (1989), 634-637. Another possible structural change can be the formation of Triple strands, such as B. homopyrimidine nucleic acid, include. This is good in the article by P. Rajagopal et al. in Nature, 339 (1989), 637-640.

Fragments comprising the MS 51 mini satellite and Hinf I fragments in particular can therefore be used as control fragments be useful in the method according to the invention. It it is of course pointed out that the MS 51 mini satellite not only when determining sample DNA fragmentation is useful, but also, for example, as a single locus probe in methods of genetic characterization, as described for example in EU-OS 2 38 329.

The MS 51 mini satellite is preferred by the following nucleotide sequence designated pMS 51, namely

5′-ACATGGCAGG (AGGGCAGG) _n TGGAGGG-3 ′,

where n = 1 or 2, and tandem repeats thereof identifiable. Any convenient number of repetitions can be present.

Accordingly, the invention relates to in another respect Polynucleotide that selectively attaches to a sample DNA fragment hybridizes, which is specifically by the nucleotide sequence

5′-ACATGGCAGG (AGGGCAGG) _n TGGAGGG-3 ′,

where n = 1 or 2, and tandem repeats thereof are identifiable. The sample DNA fragment expediently results from a restriction enzyme cutting, for example a Hinf I cutting, of, for example, DNA. It should be noted that the tandem repeats are not necessarily perfect repeats of the given frequency, provided that there is a reasonable degree of sequence homology.

In a preferred aspect, the present relates to Invention a polynucleotide, which is the most immediate nucleotide sequence and tandem repeats given above and complementary sequences or any Part of it with, for example, at least 6, at least 8 or preferably contains at least 10 nucleotides. The nucleotide includes in particular the nucleotide sequence and any convenient number of repetitions of it.

Finally, the invention also relates to probes which expediently together one of the above polynucleotides contain with a labeling or marking component, to make it easier to find them.

The invention also relates to a fragmentation control kit for the determination of a control fragment. The The kit expediently comprises a mini satellite probe, which selectively hybridizes to a control fragment that Sample DNA digestion with Hinf I after the formation of an or formed several of the DNA fragments to be characterized becomes. At least one of the following components is in the kit also before: a suitable buffer, packaging and one Instructions for use. If necessary, the kit can also do that Restriction enzyme Hinf I included.

According to a preferred embodiment, the Control kit a polynucleotide probe that selectively attaches to a Sample DNA fragment hybridized specifically by the Nucleotide sequence

5′-ACATGGCAGG (AGGGCAGG) _n TGGAGGG-3 ′,

where n = 1 or 2, and tandem repeats thereof are identifiable. The sample DNA fragment expediently results from the restriction enzyme cutting, for example cutting with Hinf I, a sample DNA.

In a more preferred embodiment, the Control kit a polynucleotide probe which is the immediate nucleotide sequence and tandem repeats given above thereof and sequences complementary thereto. The Sample DNA fragmentation agent is preferably a Restriction enzyme and especially Hinf I.

The invention will now be described with reference to the drawings and Examples explained in more detail.

Fig. 1 shows the results of partial digestion of a genomic DNA sample. The sample was digested for 20 min, 40 min, 60 min, 1 h, 2 h, 4 h and 24 h, respectively. The bands clearly show how the DNA fingerprints change over time.

Fig. 2 shows the results of a probe of the same DNA sample with pMS 51. The bands can clearly recognize the resilience of MS 51 alleles relative to a digestion and the change pattern of the bands.

Fig. 3 shows the results of probing the same DNA sample with the probe p Lambda g3. It can be seen that the g3 alleles show no appreciable level of fragmentation resistance and that the bands stabilize quickly. It should be noted that only one allele is visible when using this sample.

Figure 4 shows the location of the Eco RI, Hind III, Hinf I, Pst I and Sau 3A restriction sites in the vicinity of the MS 51 mini satellite site.

In the following examples, DNA isolation, the Restriction enzyme digestion, electrophoresis and that Southern blotting is carried out as follows:

DNA isolation

DNA was made from the whole blood of a human donor (AFM 16) isolated. White blood cells were made from 20 ml of blood separated and once in 1xSSC (20xSSC is 3.0 M NaCl / 0.3 M Trisodium citrate). The cells were then placed in 3.75 ml 0.2 M sodium acetate pH 7.0, 100 µl 10 mg / ml Proteinase-K (Boehringer-aus-Tritirachiumalbum) and 250 µl 10% SDS at 56 ° lysed for 3 h. The lysate was then treated with phenol / chloroform (25 parts phenol; 24 parts chloroform; 1 part isoamyl alcohol) and once with chloroform (24 parts chloroform; 1st Part isoamyl alcohol) extracted.

DNA was added after adding 2 volumes of 100% ethanol unwrapped and again solvated in 0.2 M sodium acetate. they was then precipitated with 100% ethanol, in 80% ethanol rinsed and then dried. In conclusion, the DNA was back completely solvated in 10 mM Tris pH 8/1 mM EDTA buffer.

Restriction enzyme digestion

The concentration of the DNA was determined by fluorimetry Aliquots of Hinf I digested basic DNA using a Hoeffer T 120-100 DNA fluorimeter. 30 µg one high molecular weight DNA extracted from AFM 16 blood was supplied in a volume of 140 μl with Hinf I enzyme ( digested by BCL with 20 units / µl). 140 units Hinf I- Enzyme were used and incubated at 37 ° C, with in Intervals of 10 min during the first hour of incubation was mixed.

20 ul aliquots were placed in 1 ul 100 mM EDTA pH 8 and after 20, 40 and 60 min as well as after 2, 3, 4 and 24 h frozen.  

Electrophoresis

A 0.7% 20 cm × 25 cm agarose gel (Sigma-Low EEO Type I) was in 1 x TBE (134 mM Tris, 74.9 mM H₃BO₃, 2.5 mM Na₂ EDTA pH 8) electrophoresis buffer prepared. The Hinf I restricted DNA samples were taken along with molecular weight markers and 0.5 µg pBR 322 plasmid DNA (Anglian Biotechnology, 2 mg / ml), those with Bg1I restriction enzyme (supplied by Anglian Biotechnology had been digested with 10 units / µl), upset. A constant voltage of 75 V was found on Agarose gel applied, and the 2.3-kilobase molecular weight marker from the pBR 322 Bg1I DNA was 18.5 cm from the origin let run.

Southern blotting

The gel was depurinated in 0.25 M HCl for 15 min, in 30 min 0.5 M NaOH / 1.5 M NaCl denatured and in 0.5 Tris pH 7.5, 3.0 M NaCl neutralized for 30 min. The DNA was through Southern blotting transferred to Hybond-N (Amersham) and by UV radiation fixed.

Example 1 Probe labeling and hybridization

80ng 33.15 probe (manufactured according to that in GB-PS 21 66 445 described procedures) were carried out with alpha 32 P dGTP (DuPont - 3000 Ci / mmol in 10 mM Tricine) characterized by primer extension.

36 ul buffer B was added to 24 ul 33.15 buffer and 5 min cooked and then cooled on ice for 10 min. 16 µl Buffer C was then mixed with 20 ul alpha 32 P dGTP and 4 ul Klenow enzyme (BCL sequencing grade 5 units / µl) added. The reaction was incubated for 3 hours at room temperature. This is sufficient probe for a hybridization chamber (a lockable Perspex box measuring 21 cm × 21 cm).

A NICK column (Pharmacia-Sephadex G-50 column) was used 10 ml column buffer (20 mM Tris pH 7.5, 20 mM NaCl, 2 mM EDTA, 0.25% SDS). The probe reaction mixture was passed through the column, 0.4 ml  Column buffers were then added and drained another 0.4 ml column buffer was added, and this was collected and used as a cleaned probe.

The Hybond-N filter was moistened in 1 x SSC and in 1 x Denhardts (0.2% BSA) (Sigma Fraction V), 0.2% Ficoll 400, 0.2% polyvinyl pyrolidone (average molecular weight 40,000, 1 x SSC) prehybridized at 62 ° C for 60 min.

0.5 ml 1 mg / ml sheared human placenta DNA (sheared in 0.3 M NaOH, 20 mM EDTA at 100 ° C for 5 min and then neutralized with HCl) were added to the probe to be used added via the hybridization chamber and boiled for 7 min and then chilled on ice for 10 min.

The probe was made into 160 ml CHFM (6% polyethylene glycol 6000 1 x Denhardts, 0.1% SDS, 6% polyethylene glycol 6000) in added to a prehybridization chamber. The filter was out transferred to the prehybridization solution and 16 h at 62 ° C incubated with moderate shaking.

The filter was washed with 400 ml 1 x SSC, 25 ug / ml herring sperm DNA (Sigma), 0.1% SDS washed at 62 ° C for 30 min, which was four times was repeated. It was then rinsed in 3 x SSC and briefly on air dried before being wrapped in a Saran wrap has been. He was then in a Curix MR 800 Intensifying sieve (Agfa) in a light-tight cassette introduced and then for 10 days at -70 ° C on a for X-ray sensitive film exposed.

Buffer B:
44 µl buffer A (1.21 M Tris pH 8.0, 0.12 M MgCl₂, 0.25 M 2-mercaptoethanol), 110 µl 2 m Hepes pH 6.6, 242 µl MilliQ H₂O (water system of the Millipore reagent Variety), aliquoted and stored at -20 ° C.

33.15 buffer:
20 µl Diag 25 7.7 µl (custom prepared oligonucleotides) 4 µl 20 ng / µl 33.15 insert.

Buffer C:
44 µl, 1 mM dATP, 44 µl 1 mM dCTP, 44 µl 1 mM TTP and 44 µl 10 mg / ml BSA, aliquoted and stored at -20 ° C (dATP, dCTP, TTP-Pharmacia 100 mM solutions).

Example 2 Probe labeling and hybridization

20 ng of the probe MS 51 (the insert sequence is in this Description given) were by random oligonucleotide priming (Feinberg and Vogelstein, 1984, Anal. Biochem., 137, Pages 266-267). The probe was run for 5 min cooked and then cooled on ice for 10 min. 10 µl Oligolabelling buffers were mixed with 4 µl 5 mg / ml Bovine serum albumin (BRL), 7 µl alpha 32 P dGTP (DuPont) and 2 µl Klenow enzyme (Boehringer sequencing variety with 5 units / µl) admitted. The reaction volume was determined by adding MilliQ H₂O brought to 50 ul and mixed and 3 h at room temperature incubated. The probe was cleaned by a NICK column, as indicated in Example 1 above. The Hybond-N filter was stripped from the 33.15 probe of Example 1 by washing in 0.4 M NaOH at 45 ° C for 30 min, then in 0.1 x SSC, 0.1 x SDS, 0.2 M Tris pH 7.5 for 30 min at 45 ° C neutralized.

The filter was then rinsed in 1 x SSC and in at 20 ° C for 20 min 0.1% bovine serum albumin (Bethesda Research Laboratories- Nucleic acid enzyme variety), 1mM EDTA, 7% SDS, 0.5M Sodium pyrophosphate pH 7.2 (Church and Gilbert, 1984, P.N.A.S., USA, 81, pages 1991-1995) prehybridized. The cleaned probe was sheared with 0.5 ml of 1 mg / ml for 5 min human placenta DNA cooked, then in ice for 10 min cooled and to 160 ml 1 x SSC, 6% Polyeturethanglycol 6000, admitted. The filter was transferred from the hybridization solution and incubated at 65 ° C for 16 h. After hybridization the filters at 65 ° C in 40 mM sodium phosphate, 0.1% SDS (pH 7.2) washed for 15 min and then twice during 15 min at 65 ° C in 0.5 x SSC, 0.01% SDS and twice 15 min at Washed 65 ° C in 0.2 x SSC, 0.01 SDS. The filter was then in  3 x SSC rinsed, briefly air-dried and in a Saran coating wrapped up for autoradiography as it is in Example 1 is described, however, the exposure time 1 was up to 2 days.

Example 3

The materials and methods were exactly the same as in Example 2, except that the probe p Lambda g3 (manufactured according to the procedures described in EU-OS 2 38 329) instead of pMS 51 was used. The sequence of p lambda is g3

AGGAATAGAAAGGCGGGYGGTGTGGGCAGGGAGRGGC

(where Y = C, T or U, R = A or G and T = T or U) and tandem repeats thereof.

Claims (12)

1. method for determining a sample DNA fragmentation, in which the presence of a control fragment is determined that is during sample DNA cutting after formation one or more of the DNA fragments to be characterized is formed. 2. The method of claim 1, wherein the sample DNA is fragmented by enzymatic digestion. 3. The method according to claim 1 or 2, wherein the Control fragment a complete sample DNA fragmentation displays. 4. The method according to any one of claims 1 to 3, in which the control fragment using a mini satellite probe is determined. 5. The method of claim 4, wherein the mini satellite probe used to be a single mini satellite area or hypervariable location. 6. The method of claim 5, wherein the minisatellite probe selectively hybridizes to a sample DNA fragment that is specifically identified by the nucleotide sequence 5'-ACATGGCAGG (AGGGCAGG) _n TGGAGGG-3 ', where n = 1 or 2, and tandem repeats thereof is. 7. The method according to any one of claims 1 to 6, in which the restriction enzyme Hinf I used to cut the sample DNA becomes. 8. Polynucleotide which hybridizes selectively to a sample DNA fragment which is specifically identified by the nucleotide sequence 5'-ACATGGCAGG (AGGGCAGG) _n TGGAGGG-3 ', where n = 1 or 2, and tandem repeats thereof. 9. The polynucleotide of claim 8, together with a Labeling or marking component. 10. Polynucleotide probe containing the nucleotide sequence 5'-ACATGGCAGG (AGGGCAGG) _n TGGAGGG-3 ', where n = 1 or 2, and tandem repeats thereof. 11. Control kit, which is a mini satellite probe, which selectively hybridizes to a control fragment that Sample DNA digestion with Hinf I after formation of a or formed several of the DNA fragments to be characterized and at least one buffer and / or packaging and / or contains instructions for use. 12. The control kit of claim 11, wherein the mini-satellite probe contains the nucleotide sequence 5'-ACATGGCAGG (AGGGCAGG) _n TGGAGGG-3 ', wherein n = 1 or 2, and tandem repeats thereof.