Diagnosis of haemophilia B using the polymerase chain reaction

The polymerase chain reaction (PCR) was used to amplify specific DNA sequences within the factor IX gene of haemophilia B patients and their relatives. Three of the amplified fragments contain polymorphic sites, which can be used as markers in segregation analyses. These restriction fragment length polymorphisms (RFLPs) were until recently detected by Southern blotting after digestion with the restriction enzymes Taq I, Dde I and Xmn I. All three RFLP's are located in introns of the factor IX gene and together are informative in approximately 70% of all cases. Each of the polymorphisms was successfully used in carrier detection studies after amplification of the relevant fragments. This method is also suitable for rapid antenatal diagnosis. Additionally we were able to amplify all eight exons of the factor IX gene including the splice junctions and a part of the 5'-region. Large deletions or insertions can be detected without further analysis. Several possibilities for the rapid detection of point mutations after DNA amplification have been described recently. The complete amplification of all functional parts of the Factor IX gene in combination with these new techniques should enable us to detect the majority of mutations leading to haemophilia B.     

A DNA marker closely linked to the factor IX (haemophilia B) gene

Summary We have isolated a DNA segment, pX58dIIIc, from an X-chromosome library which identifies an SstI restriction fragment length polymorphism (RFLP) at locus DXS99. Linkage analysis in six informative families has shown that the DXS99 locus lies close to the factor IX gene (F9). No recombination was detected between these loci in 39 informative meioses (Z=9.79, =0.0). Therefore, DXS99 will be useful as a DNA marker for the assessment of carrier status in families with haemophilia B where intragenic markers are not informative. Heterozygosity at DXS99 is approximately 50% and, in conjunction with the RFLPs at F9, 90% of females at risk for being haemophilia B carriers should be diagnosed.     

Direct detection of point mutations by mismatch analysis: application to haemophilia B.

Rapid detection of point mutations in genomic DNA has been achieved by chemical mismatch analysis of heteroduplexes formed between amplified wild-type and target sequences in the human factor IX gene. Amplification and mismatch detection (AMD) analysis of DNA from relatives of haemophilia B patients permitted carrier diagnosis by direct identification of the presence or absence of the mutation in all cases, thus eliminating the need for the informative segregation of polymorphic markers. This extends diagnostic capability to virtually all haemophilia B families. AMD analysis permits detection of all sequence variations in genomic DNA and is therefore applicable to direct diagnosis of X-linked and autosomal diseases and for identification of new polymorphisms for genetic mapping.     

Restriction fragment length polymorphism among Israeli Holstein-Friesian dairy bulls

Israeli Holstein-Friesian dairy bulls were screened for restriction fragment length polymorphisms by hybridizing cloned DNA probes for bovine growth hormone, for chymosin, and for rat muscle beta-actin to restriction endonuclease-digested DNA immobilized on nitrocellulose filters. The population proved to be polymorphic at the growth hormone locus, with evidence consistent with the phenotypes being inherited in allelic fashion. A low level of polymorphism was also observed at one of the beta-actin gene family loci. The chymosin locus was monomorphic with the restriction enzymes utilized. The results illustrate the power of restriction fragment length polymorphism methodology in visualizing genetic variability in dairy cattle populations.     

Alphoid DNA polymorphisms for chromosome 21 can be distinguished from those of chromosome 13 using probes homologous to both.

Although alphoid DNA sequences shared among acrocentric chromosomes have been identified, no human chromosome 21-specific sequence has been isolated from the centromeric region. To identify alphoid DNA restriction fragment length polymorphisms (RFLPs) specific for chromosome 21, we hybridized human genomic DNA with alphoid DNA probes [L1.26; aRI(680),21-208] shared by chromosomes 13 and 21. We detected RFLPs with restriction enzymes ECoRI, HaeIII, MboI,StuI, and TaqI. The segregation of these RFLPs was analyzed in the 40 CEPH families. Linkage analysis between these RFLPs and loci previously mapped to either chromosome 13 or 21 revealed RFLPs that appear to be specific to chromosome 21. These polymorphisms may be useful as genetic markers of the centromeric region of chromosome 21. Different alphoid loci within the centromeric region of chromosome 13 were identified.     

Multicolour fluorescent detection and mapping of AFLP markers in chicken (Gallus domesticus)

We describe the mapping of amplified restriction fragment polymorphism (AFLP) markers in chicken (Gallus domesticus) using a multi-colour fluorescent detection system. DNA was used from a population consisting of four families with a total of 183 F2 individuals. The enzyme combination EcoRI/TaqI was used for double digestion, and fluorescently labelled fragments were analysed on an ABI PRISM 377 DNA sequencer. Polymorphic signals in the range of 50-500 bp were genotyped with the ABI PRISM Genotyper 2.0 software, which enabled the analysis of both dominant and incomplete dominant markers (with respect to AFLP, often referred to as codominant). In 19 sets consisting of 3 EcoRI/TaqI primer pair combinations each, a total of 475 polymorphic markers was detected. From these polymorphisms 344 markers could be mapped on the Wageningen linkage map. Fourteen markers were length polymorphisms of the same fragment and 28 markers Z-linked and uniformative; 64 AFLP markers appeared to be unlinked and 25 AFLP markers could not be accurately mapped on the basis of the genotyping results. The resulting AFLP/microsatellite linkage map is comprised of 33 linkage groups with a total of 835 loci.     

Intrinsic polymorphism of variable number tandem repeat loci in the human genome.

In the human genome, short tandem repetitive (STR) DNA sequences often show restriction fragment length polymorphisms (RFLPs) due to variation in the number of copies of the repeat unit. For a subset of these sequences known as minisatellites or variable number tandem repeat loci (VNTR), it has been proposed that a homologous "core" sequence of 10-12 nucleotides is involved in the mechanism(s) generating the polymorphism. In our present study we have prepared oligonucleotide probes complementary to one or two repeat units of several VNTR loci. Under stringent hybridization and wash conditions these probes hybridize locus specifically thus allowing the evaluation of the intrinsic polymorphism of individual loci. Our results indicate that not all of the loci having STR DNA sequences are polymorphic despite the fact that they share the "core" sequence. This suggests that more than the DNA sequence of the locus is involved in the mechanism(s) generating the polymorphism.     

A new MspI restriction fragment length polymorphism in the hemophilia B locus

Summary Using a partial cDNA probe for human coagulation factor IX, we have detected a new restriction fragment length polymorphism in human DNA digested with MspI. The frequency of the minor allele is 0.20±0.05 and average heterozygosity is about 0.32. The MspI RELP is in strong linkage disequilibrium with the TaqI RFLP previously described, but should nevertheless be useful in segregation analysis in case of homozygosity for the TaqI minor allelc.     

A DNA probe detecting multiple haplotypes of the human Y chromosome.

We have characterized a DNA probe (49f) that detects about 15 Y-specific TaqI bands corresponding to a low-copy number sequence. Five of these bands, each representing a single DNA fragment, can either be present, absent, or variable in length. Familial segregation studies have shown that the variations of these fragments are inherited in a Mendelian fashion and strictly Y-linked. A survey of 44 male individuals indicated that the five variable TaqI fragments detected by probe 49f can be considered as five independent allelic series. Each series represents the different and mutually exclusive allelic forms observed for a single DNA fragment. A total of 16 haplotypes, each defined by a different combination of the various forms of each of these five restriction fragment length polymorphisms, were observed among the 44 scored individuals. These TaqI restriction polymorphisms are not observed with other restriction digests and have therefore been attributed to point mutations. The five polymorphic fragments map to Yq11, a region that does not recombine with the X chromosome and are therefore not redistributed. This implies that an apparently independent reassortment of one of these series with respect to the others can be explained only on the basis of mutations that occurred several times (or reverted) during evolution of the Y chromosome. However, an examination of the different combinations of two or more allelic series suggests that some alleles are not randomly distributed and raises the possibility of establishing a genealogy of the human Y chromosome.     

DNA分子标记在实验动物遗传分析中的应用

DNA分子标记技术很多,基本都是建立在RFLP、PCR和重复顺序的基础上的。本文重点介绍了限制性片段长度多态性（RFLP）标记、随机扩增多态性DNA（RAPD）标记、微卫星DNA（STR）标记、DNA指纹（DFP）标记、扩增片段长度多态性（AFLP）标记等几种重要的DNA分子标记技术的定义、结构、分布、组成、保守性、优点及丰富的多态性等。并重点介绍了微卫星DNA（STR）标记在分子遗传监测、遗传多样性分析和遗传血缘关系及个体识别等领域的应用。     

DNA fingerprinting of human cell lines using PCR amplification of fragment length polymorphisms

Summary Methods for monitoring cell line identification and authentication include species-specific immunofluorescence, isoenzyme phenotyping, chromosome analysis, and DNA fingerprinting. Most previous studies of DNA fingerprinting of cell lines have used restriction fragment length polymorphism analysis. In this study, we examined the utility of an alternative and simpler method of cell line DNA fingerprinting—polymerase chain reaction (PCR) amplification of fragment length polymorphisms. Fourteen human cell lines previously found by other methods to be either related or disparate were subjected to DNA fingerprinting by PCR amplification of selected fragment length polymorphism loci. Cell identification patterns by this method were concordant with those obtained by isoenzyme phenotyping and restriction fragment length polymorphism-DNA fingerprinting, and were reproducible within and between assays on different DNA extracts of the same cell line. High precision was achieved with electrophoretic separation of amplified DNA products on high resolution agarose or polyacrylamide gels, and with fragment length polymorphism (FLP) loci-specific “allelic ladders” to identify individual FLP alleles. Determination of the composite fingerprint of a cell line at six appropriately chosen fragment length polymorphism loci should achieve a minimum discrimination power of 0.999. The ability of PCR-based fragment length polymorphism DNA fingerprinting to precisely and accurately identify the alleles of different human cell lines at multiple polymorphic fragment length polymorphism loci demonstrates the feasibility of developing a cell line DNA fingerprint reference database as a powerful additional tool for future cell line identification and authentication.     

Restriction maps and restriction fragment length polymorphisms of the human 21-hydroxylase genes

Restriction maps were constructed for the two human 21-hydroxylase genes (21-OHA and 21-OHB) by using DNA from subjects homozygous for a deletion of each gene. Comparing the patterns of these two genes, a KpnI restriction site occurred in the 21-OHA gene in place of a TaqI site in the 21-OHB gene about 1-kb from the 5' end of the gene, and an extra EcoRI site was located 500 bp 5' to the common EcoRI site. The DNA of fourteen unrelated normal subjects was digested with nine restriction endonucleases (AccI, BamHI, BgIII, EcoRI, HindIII, KpnI, MspI, SacI and TaqI). Restriction fragment length polymorphisms were found with EcoRI, HindIII and AccI that resulted from polymorphic endonuclease sites outside the genes.     

DNA sequence variation within the beta-glucuronidase gene complex among inbred strains of mice

Tightly linked to the gene that encodes murine beta-glucuronidase (GUS) are three GUS-specific regulatory elements. Together, these elements define the GUS gene complex. Specific alleles of each regulatory element are associated with a specific GUS structural allele. These associations define the three common forms (haplotypes) of the GUS gene complex, designated A, B, and H. As an initial step in defining the DNA determinants of each regulatory element and to develop DNA markers for the common haplotypes, we have identified several DNA variants by blot hybridization analysis of restricted genomic DNA using GUS-specific cDNA probes. Of 30 tested restriction endonucleases, 24 reveal DNA polymorphisms that distinguish B- and H-haplotype DNA from that of the A haplotype. Of these 24, 18 uncover a restriction fragment length polymorphism in which the polymorphic fragment of A-haplotype DNA is 200-300 bp larger than the corresponding fragment of B- or H-haplotype DNA. DNA sequence analysis of this polymorphic region reveals the presence of a short, interspersed repetitive element of the B2 family within A-haplotype DNA which is absent in DNAs of B- or H-haplotype mice. None of the DNA variations revealed by these analyses can be associated at this time with variation in the regulatory or structural properties of GUS among the common haplotypes. Nevertheless, they do provide useful haplotype-specific markers within the GUS gene complex which are of critical importance for DNA transfer experiments in transgenic mice and in cultured cells.     

MspI polymorphic site within the Factor IX gene

Summary We have localized the position of the MspI polymorphic site that exists within the factor IX gene. The location of the MspI polymorphic site is within intro D, 1.9 kb upstream from the beginning of exon V and 4 kb downstream from a known polymorphic TaqI site. The use of a specific genomic probe simplifies the interpretation of the MspI polymorphism by reducing the number of non-overlaping DNA fragments to three bands; 2.4, 3.4, and 5.8 kb.     

Restriction fragment length polymorphism of the rat albumin gene in Sprague-Dawley rats and its application in genetic study of analbuminemia.

Restriction fragment length polymorphism of the rat albumin gene was discovered in a stock of Sprague-Dawley rats by Southern blots of rat liver DNAs using cloned albumin cDNA, prAlb-1 (1), as a probe. The polymorphic DNA fragments were observed when rat DNAs were digested with either Hind III or Pst 1 and the difference in length of the DNA fragments in Hind III or Pst 1 digests was estimated as 1.4 kbp. When DNAs were digested with EcoR I, restriction fragment length polymorphism was not observed. Therefore, this polymorphic DNA was concluded to be located in the flanking sequence. Structural analysis of the cloned albumin gene showed that the polymorphism was located in the 3'-flanking sequence. With this polymorphism as a marker of the albumin structural gene, the phenotype of analbuminemia, which is an autosomally recessive trait, was found to be linked to the structural gene of albumin.     

Restriction sites containing CpG show a higher frequency of polymorphism in human DNA

Unique loci in the human genome were examined with restriction enzymes in order to detect restriction fragment length polymorphisms (RFLPs). Of 31 arbitrary loci, nine were detectably polymorphic, reflecting ten polymorphic restriction sites. Nine of the ten polymorphic sites were revealed with two restriction enzymes, Msp I and Taq I, whose recognition sequences have in common the dimer sequence CpG. The cytosines in the CpG sequence are known to be frequently methylated in mammals, and the occurrence of significant variation in Msp I and Taq I sites supports the view that methylated cytosine residues are hotspots for mutation in mammalian DNA.     

Linkage studies of polymorphic, repeated DNA sequences in centromeric regions of human chromosomes.

The DNA at human centromeric regions was characterized by using a repetitive sequence, 308, which localizes in situ exclusively to centromeres of all chromosomes. We previously noted that this sequence is enriched on chromosome 6 and has chromosome-specific organization on 6, 3, 7, 14, X, and Y. In addition to this basic organization, sequences homologous to 308 are polymorphic among normal individuals. The variants are transmitted in a Mendelian manner within a family. To determine the chromosome origin of the variants, we studied their linkage to markers of various chromosomes. Linkage analysis of one pedigree segregating two polymorphisms shows that the 2.6-kilobase (kb) BamHI and 2.6-kb TaqI fragments are linked to each other and to the HLA loci on chromosome 6. Data from another family shows that 2.8-kb TaqI, 4.0-kb TaqI, and 1.3-kb BamHI polymorphic fragments are linked and are probably near the Fy locus on chromosome 1. By dot blot analysis, we determined that the relative amount of these sequences in the genome is not measurably different between unrelated individuals. Thus, the polymorphisms represent changes in homologous 308 sequences on specific chromosomes and can be used as chromosome-specific markers. Linkage studies using polymorphisms of repeated sequences will be most useful within a kindred, especially from an inbred population, because polymorphic repeats of the same restriction size may be heterogeneous in origin.     

Restriction fragment length polymorphisms associated with factor VIII: C gene in Chinese

Summary Twenty-six unrelated hemophilia A and 70 unrelated normal chromosomes in 184 subjects were studied to determine the frequencies of intragenic and intergenic restriction fragment length polymorphisms associated with the factor VIII:C gene. The incidences for positive BclI and BglI polymorphic sites in the Chinese were 82% and 100%, respectively. Both were higher than in other ethnic groups, while the incidence for XbaI polymorphism was 57%, which is similar to that reported in Caucasians. Using the St 14.1 probe, two polymorphic TaqI allelic systems in the DXS52 region were detectable, with heterozygous rates of 0.712 (for system I, alleles 1 to 8) and 0.495 (for system II, and alleles), respectively. Thus, using a combination of four polymorphisms, it would be possible to offer carrier detection or prenatal diagnosis in 96% of Chinese females at risk.     

Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site.

DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5' end of intron d and the two last coding nucleotides located at the 3' end of exon IV in the normal factor IX gene; this fragment has been replaced by a 47-bp sequence from the normal factor IX gene, although this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.     

The polymorphic locus for glycogen storage disease VI (liver glycogen phosphorylase) maps to chromosome 14.

Human liver glycogen phosphorylase deficiency, also known as glycogen storage disease type VI (GSD VI) or Hers disease, is characterized by hepatomegaly and reduced or absent glycogenolytic response to the injection of glucagon. The recently isolated cDNA encoding the liver isozyme of glycogen phosphorylase was used to map the gene and identify restriction-fragment polymorphisms in normal Caucasians as a prerequisite for detecting linked GSD VI abnormalities. Results of restriction-enzyme analysis using a downstream fragment of the liver glycogen phosphorylase cDNA indicated the existence of a single gene copy per haploid genome. Hybridization of this downstream liver phosphorylase probe to dual laser-excited, sorted human chromosomes localized the gene to human chromosome 14. When the downstream probe was tested on genomic DNA cut with seven different restriction enzymes, a single MspI restriction-fragment-length polymorphism (RFLP) was observed in a single individual. In contrast, similar Southern blots performed with an upstream portion of the cDNA encoding liver phosphorylase revealed common RFLPs for four of eight enzymes tested, with minor polymorphic allele frequencies ranging from 33% to 44%. One of the four enzymes (TaqI) revealed two independent polymorphisms. If random distribution of these haplotypes among normal and disease loci, is assumed, approximately 92% of fetuses at risk for Hers disease will be informative when tested with the upstream liver phosphorylase probe.