Process for the detection of restriction fragment length polymorphisms in eukaryotic genomes

Claim 2

In a process for the determination of the genetic interrelationships of individuals comprising analysis of eukaryotic DNA wherein DNA of an individual is isolated, the DNA is subjected to restriction endonuclease digests, the cleaved DNA is separated by gel electrophoresis, the gel is dried and hybridized with a probe, and the pattern of the DNA is evaluated and compared to that of other individuals, the improvement which comprises use of a probe which comprises a chemically synthesized oligonucleotide which specifically hybridizes to eukaryotic simple tandem repeats having a periodicity of 2 to 4 bases.

Claim 3

The process according to claim 1, characterized in that the DNA used is human or animal DNA.

Claim 4

The process according to any one of claims 1 and 2, characterized in that the restriction enzyme used is Alu I, Hae III, Hinf I, or Mbo I.

Claim 5

The process according to claim 1, characterized in that the probe used is an oligonucleotide which specifically hybridizes to eukaryotic simple quadruplet repeats (sqr).

Claim 6

The process according to claim 4, characterized in that the simple quadruplet repeats (sqr) recognized by the oligonucleotide probe in the eukaryotic genome are GA A sequences or their complement sequences T TC.

Claim 7

The process according to claim 5, characterized in that the oligonucleotide has a length of 16 to 23 nucleotides.

Claim 8

The process according to claim 6, characterized in that the oligonucleotide is (GATA) 4 GA or its complement sequence TC(TATC) 4 .

Claim 9

The process according to claim 6, characterized in that the nucleotide is a 20-mer.

Claim 10

The process according to claim 6, characterized in that the oligonucleotide is (GATA) 5 or its complement sequence (TATC) 5 .

Claim 11

The process according to claim 6, characterized in that t he oligo nucleotide is (GATA) 2 GACA(GATA) 2 or its complement sequence (TATC) 2 TGTC(TATC) 2 .

Claim 12

The process according to claim 6, characterized in that the oligonucleotide is (GATA) 4 , (GACA) 4 , or their complement sequences (TATC) 4 or (TGTC) 4 .

Claim 13

The process according to claim 6, characterized in that the oligonucleotide is (GGAT) 4 or its complement sequence (ATCC) 4 .

Claim 14

The process according to claim 1, characterized in that the probe used is an oligonucleotide which specifically hybridizes to eukaryotic simple triplet repeats.

Claim 15

The process according to claim 13, characterized in that the simple triplet repeats recognized by the oligonucleotide probe in the eukaryotic genome are TCC sequences or their complement sequences GGA.

Claim 16

The process according to claim 14, characterized in that the oligonucleotide has a length of 15 to 24 nucleotides.

Claim 17

The process according to claim 15, characterized in that the oligonucleotide is (TCC) 5 or its complement sequence (GGA) 5 .

Claim 18

The process according to claim 1, characterized in that the probe used is an oligonucleotide which specifically hybridizes to eukaryotic simple doublet repeats.

Claim 19

The process according to claim 17, characterized in that the simple duplet repeats recognized by the oligonucleotide probe in the eukaryotic genome are GT sequences or their complement sequences AC.

Claim 20

The process according to claim 18, characterized in that the oligonucleotide has a length of 16 to 24 nucleotides.

Claim 21

The process according to claim 19, characterized in that the oligonucleotide is (GT) 8 or its complement sequence (AC) 8 .

Claim 22

The process according to claim 19, characterized in that the oligonucleotide is (CT) 8 or its complement sequence (AG) 8 .

Claim 23

The process according to claim 1, characterized in that the consecutive hybridization and evaluation operation comprises hybridizing the cleaved DNA with the radioactively labeled probe, removing the unhybridized part of the probe, exposing an X-ray film to the hybridized product, removing the hybridized product from the gel and repeating the operation with another probe.

Claim 24

The process according to claim 1, characterized in that the hybridization and evaluation operation comprises transferring the cleaved DNA onto a solid support, hybridizing the DNA with the biotinylated probe and developing the hybridized product with strepdavidin.

Claim 25

The process according to claim 6, wherein the oligonucleotide has one of the following sequences: ______________________________________ (a) oligonucleotide (GATA).sub.4 GA ATA(GATA).sub.3 GAG TA(GATA).sub.3 GAGA A(GATA).sub.3 GAGAT (GATA).sub.3 GA(GATA) ATA(GATA).sub.2 GA(GATA)G TA(GATA).sub.2 GA(GATA)GA A(GATA).sub.2 GA(GATA)GAT (GATA).sub.2 GA(GATA).sub.2 ATA(GATA)GA(GATA).sub.2 G TA(GATA)GA(GATA).sub.2 GA A(GATA)GA(GATA).sub.2 GAT (GATA)GA(GATA).sub.3 ATAGA(GATA).sub.3 G TAGA(GATA).sub.3 GA AGA(GATA).sub.3 GAT GA(GATA).sub.4 A(GATA).sub.4 G (GATA).sub.4 GA or its complement sequence TC(TATC).sub.4 C(TATC).sub.4 T (TATC).sub.4 TC ATC(TATC).sub.3 TCT TC(TATC).sub.3 TCTA C(TATC).sub.3 TCTAT (TATC).sub.3 TC(TATC) ATC(TATC).sub.2 TC(TATC)T TC(TATC).sub.2 TC(TATC)TA C(TATC).sub.2 TC(TATC)TAT (TATC).sub.2 TC(TATC).sub.2 ATC(TATC)TC(TATC).sub.2 T TC(TATC)TC(TATC).sub.2 TA C(TATC)TC(TATC).sub.2 TAT (TATC)TC(TATC).sub.3 ATCTC(TATC).sub.3 T TCTC(TATC).sub.3 TA CTC(TATC).sub.3 TAT TC(TATC).sub.4 b. oligonucleotide or its complement sequence (GATA).sub.5 (TATC).sub.5 ATA(GATA).sub.4 G ATC(TATC).sub.4 T TA(GATA).sub.4 GA TC(TATC).sub.4 TA A(GATA).sub.4 GAT C(TATC).sub. 4 TAT (GATA).sub.5 (TATC).sub.5 c. oligonucleotide ATA(GATA)GACA(GATA).sub.2 G TA(GATA)GACA(GATA).sub.2 GA A(GATA)GACA(GATA).sub.2 GAT (GATA)GACA(GATA).sub.3 ATAGACA(GATA).sub.3 G TAGACA(GATA).sub.3 GA AGACA(GATA).sub.3 GAT GACA(GATA).sub.4 ACA(GATA).sub.4 G CA(GATA).sub.4 GA A(GATA).sub.4 GAC (GATA).sub.4 GACA ATA(GATA).sub.3 GACAG TA(GATA).sub.3 GACAGA A(GATA).sub.3 GACAGAT (GATA).sub.3 GACA(GATA) ATA(GATA).sub.2 GACA(GATA)G TA(GATA).sub.2 GACA(GATA)GA A(GATA).sub.2 GACA(GATA)GAT (GATA)GACA(GATA).sub.2 or its complement sequence (TATC).sub.2 TGTC(TATC).sub.2 ATC(TATC)TGTC(TATC).sub.2 T TC(TATC)TGTC(TATC).sub.2 TA C(TATC)TGTC(TATC).sub.2 TAT (TATC)TAGTC(TATC).sub.3 ATCTGTC(TATC).sub.3 T TCTGTC(TATC).sub.3 TA CTGTC(TATC).sub.3 TAT TGTC(TATC).sub.4 GTC(TATC).sub.4 T TC(TATC).sub.4 TG C(TATC).sub.4 TGT (TATC).sub.4 TGTC ATC(TATC).sub.3 TGTCT TC(TATC).sub.3 TGTCTA C(TATC).sub.3 TGTCTAT (TATC).sub.3 TGTCTATC ATC(TATC).sub.2 TGTCTATCT TC(TATC).sub.2 TGTCTATCTA C(TATC).sub.2 TGTCTATCTAT (TATC).sub.4 TGTC(TATC).sub.2 (d) oligonucleotide or its complement sequence (GATA).sub.4 (TATC).sub.4 ATA(GATA).sub.3 G ATC(TATC).sub.3 T TA(GATA).sub.3 GA TC(TATC).sub.3 TA A(GATA).sub.3 GAT C(TATC).sub.3 TAT (GATA).sub.4 (TATC).sub.4 (GACA).sub.4 (TGTC).sub.4 ACA(GACA).sub.3 G GTC(TGTC).sub.3 T CA(GACA).sub.3 GA TC(TGTC).sub.3 TG A(GACA).sub.3 GAC C(TGTC).sub.3 TGT (GACA).sub.4 (TGTC).sub.4 (e) oligonucleotide or its complement sequence (GGAT).sub.4 (ATCC).sub.4 GAT(GGAT).sub.3 G TCC(ATCC).sub.3 A AT(GGAT).sub.3 GG CC(ATCC).sub.3 AT T(GGAT).sub.3 GGA C(ATCC).sub.3 ATC (GGAT).sub.4 (ATCC).sub.4 - ______________________________________

Claim 26

The process according to claim 15, wherein the oligonucleotide is (CCT) 5 or (CTC) 5 or their complement sequences (AGG) 5 or (GAG) 5 , respectively.

Claim 27

The process according to claim 19, wherein the oligonucleotide is (TG) 8 or its complement sequence (CA) 8 or is (TC) 8 or its complement sequence (GA) 8 , respectively.