Individual-specific 'fingerprints' of human DNA

Simple tandem-repetitive regions of DNA (or 'minisatellites') which are dispersed in the human genome frequently show substantial length polymorphism arising from unequal exchanges which alter the number of short tandem repeats in a minisatellite. We have shown previously that the repeat elements in a subset of human minisatellites share a common 10-15-base-pair (bp) 'core' sequence which might act as a recombination signal in the generation of these hypervariable regions. A hybridization probe consisting of the core repeated in tandem can detect many highly polymorphic minisatellites simultaneously to provide a set of genetic markers of general use in human linkage analysis. We now show that other variant (core)n probes can detect additional sets of hypervariable minisatellites to produce somatically stable DNA 'fingerprints' which are completely specific to an individual (or to his or her identical twin) and can be applied directly to problems of human identification, including parenthood testing.     

Minisatellite repeat coding as a digital approach to DNA typing

Most DNA typing systems used in forensic and legal medicine assay allelic length variation at tandem repetitive DNA regions such as minisatellites. A simple alternative approach that displays patterns of variant repeat units along minisatellite alleles is described here. This produces DNA profiles as extraordinarily variable digital sequences appropriate for forensic investigations, including computer databasing, and for analysing allele diversity and the role of recombination in minisatellite instability.     

Hypervariable 'minisatellite' regions in human DNA

The human genome contains many dispersed tandem-repetitive 'minisatellite' regions detected via a shared 10-15-base pair 'core' sequence similar to the generalized recombination signal (chi) of Escherichia coli. Many minisatellites are highly polymorphic due to allelic variation in repeat copy number in the minisatellite. A probe based on a tandem-repeat of the core sequence can detect many highly variable loci simultaneously and can provide an individual-specific DNA 'fingerprint' of general use in human genetic analysis.     

Demographic study of a wild house sparrow population by DNA fingerprinting

Over the past twenty years, several techniques from biochemical and molecular genetics, such as enzyme electrophoresis and isoelectric focusing, have been widely and successfully applied to the study of population differentiation and evolution. However, they have been less applicable to demographic problems such as assigning parentage to individuals within a population. This stems from a general weakness of data derived from enzyme loci: allele frequencies at polymorphic loci are sufficiently skewed that the majority of individuals are of one or two genotypes. Many enzyme systems can only be examined post mortem, so that the loci are of little use if the animals are to be studied in the wild. The search for new and more sensitive techniques for detecting genetic variation has continued, and recently a major discovery has come from molecular biology. Jeffreys et al. have reported the detection of a type of hypervariable 'minisatellite' DNA that is extraordinarily polymorphic in human populations. We have applied their technique to several bird species and particularly to a population of house sparrows (Passer domesticus) near Nottingham. We report here that one of the human minisatellite clones is a suitable probe for sparrow DNA and that it reveals variation as extensive as that found in man. These results suggest that analysis of minisatellite DNA will be a powerful tool in the study of demographic population genetics.     

Positive identification of an immigration test-case using human DNA fingerprints

The human genome contains a set of minisatellites, each of which consists of tandem repeats of a DNA segment containing the 'core' sequence, a putative recombination signal in human DNA. Multiallelic variation in the number of tandem repeats occurs at many of these minisatellite loci. Hybridization probes consisting of tandem repeats of the core sequence detect many hypervariable minisatellites simultaneously in human DNA, to produce a DNA fingerprint that is completely individual-specific and shows somatic and germline stability. These DNA fingerprints are derived from a large number of highly informative dispersed autosomal loci and are suitable for linkage analysis in man, and for individual identification in, for example, forensic science and paternity testing. They can also be used to resolve immigration disputes arising from lack of proof of family relationships. To illustrate the potential for positive or inclusive identification, we now describe the DNA fingerprint analysis of an immigration case, the resolution of which would have been very difficult and laborious using currently available single-locus genetic markers.     

Unequal crossing over in the ribosomal DNA of Saccharomyces cerevisiae

Unequal sister chromatid exchanges occur at the ribosomal DNA locus of yeast during mitotic growth. The frequency of unequal crossing over, as measured by the deletion or duplication of an inserted genetic marker (LEU2), is sufficient to maintain the sequence homogeneity of the rDNA repeat units.     

Variable amplification of immunoglobulin lambda light-chain genes in human populations

The human lambda immunoglobulin locus displays a series of restriction fragment length polymorphisms that are readily detected in small populations of normal individuals. Similar polymorphisms appear in populations of wild mice, suggesting that the lambda locus is subject to rapid variation within a single species. Here we show that the polymorphisms seen in the human lambda locus seem to have arisen from unequal meiotic crossing over, altering the number of lambda from as few as six to as many as nine per haploid genome. This expansion and contraction in the number of human lambda genes is significant in that it may affect an individual's capacity to produce variation among lambda light chain genes.     

Conformational mutations in human mitochondrial DNA

Variation in the human mitochondrial DNA (mtDNA) sequence has been extensively analysed using restriction fragment length polymorphisms (RFLPs). MtDNA RFLPs have previously been attributed to nucleotide changes within restriction endonuclease recognition sites or to small insertion-deletion mutations. We now report that RFLPs detected by polyacrylamide gel electrophoresis can also result from single nucleotide substitutions which alter the mobility of small- to medium-sized restriction fragments that incorporate the sequence. We have defined the mutation responsible at two loci and have identified several possible additional loci. When screening human mtDNAs with multiple restriction endonucleases, such mutations can be misidentified as insertion-deletion mutations or counted as multiple polymorphic restriction sites. This can lead to errors in constructing restriction maps and estimating sequence diversity.     

应用T载体连接PCR技术分离黄嘴白鹭微卫星位点

传统的微卫星分离策略对于低丰度的物种如鸟类而言,通常是低效的.探讨了一种改良的T载体PCR序列获取法用于黄嘴白鹭(Egretta eulophotes)微卫星DNA的分离,称为T载体连接PCR(T-vector-ligation PCR,TVL-PCR).在获取第一侧微卫星序列的71个阳性克隆中,59个克隆含有重复序列,设计了48对位点特异巢式引物.经过两轮TVL-PCR后,31个位点的扩增产物为预期大小,经测序后获得21个完整的微卫星位点.除去侧翼序列过短和小卫星位点外,设计16个微卫星位点进行多态信息检测,其中7个位点显示出多态性,平均等位基因数为2～20,观察杂合度(HO)和期望杂合度(HE)分别为0.375～0.958和0.477～0.956,所有位点都符合哈迪-温伯格平衡(HWE)和连锁平衡.说明TVL-PCR技术对低丰度微卫星物种的位点分离具有一定的应用价值.     

Mutation rates differ among regions of the mammalian genome

In the traditional view of molecular evolution, the rate of point mutation is uniform over the genome of an organism and variation in the rate of nucleotide substitution among DNA regions reflects differential selective constraints. Here we provide evidence for significant variation in mutation rate among regions in the mammalian genome. We show first that substitutions at silent (degenerate) sites in protein-coding genes in mammals seem to be effectively neutral (or nearly so) as they do not occur significantly less frequently than substitutions in pseudogenes. We then show that the rate of silent substitution varies among genes and is correlated with the base composition of genes and their flanking DNA. This implies that the variation in both silent substitution rate and base composition can be attributed to systematic differences in the rate and pattern of mutation over regions of the genome. We propose that the differences arise because mutation patterns vary with the timing of replication of different chromosomal regions in the germline. This hypothesis can account for both the origin of isochores in mammalian genomes and the observation that silent nucleotide substitutions in different mammalian genes do not have the same molecular clock.     

A haplotype map of the human genome

Inherited genetic variation has a critical but as yet largely uncharacterized role in human disease. Here we report a public database of common variation in the human genome: more than one million single nucleotide polymorphisms (SNPs) for which accurate and complete genotypes have been obtained in 269 DNA samples from four populations, including ten 500-kilobase regions in which essentially all information about common DNA variation has been extracted. These data document the generality of recombination hotspots, a block-like structure of linkage disequilibrium and low haplotype diversity, leading to substantial correlations of SNPs with many of their neighbours. We show how the HapMap resource can guide the design and analysis of genetic association studies, shed light on structural variation and recombination, and identify loci that may have been subject to natural selection during human evolution.     

DNA sequence and analysis of human chromosome 8

The International Human Genome Sequencing Consortium (IHGSC) recently completed a sequence of the human genome. As part of this project, we have focused on chromosome 8. Although some chromosomes exhibit extreme characteristics in terms of length, gene content, repeat content and fraction segmentally duplicated, chromosome 8 is distinctly typical in character, being very close to the genome median in each of these aspects. This work describes a finished sequence and gene catalogue for the chromosome, which represents just over 5% of the euchromatic human genome. A unique feature of the chromosome is a vast region of approximately 15 megabases on distal 8p that appears to have a strikingly high mutation rate, which has accelerated in the hominids relative to other sequenced mammals. This fast-evolving region contains a number of genes related to innate immunity and the nervous system, including loci that appear to be under positive selection--these include the major defensin (DEF) gene cluster and MCPH1, a gene that may have contributed to the evolution of expanded brain size in the great apes. The data from chromosome 8 should allow a better understanding of both normal and disease biology and genome evolution.     

Forensic application of DNA 'fingerprints'

Many highly polymorphic minisatellite loci can be detected simultaneously in the human genome by hybridization to probes consisting of tandem repeats of the 'core' sequence. The resulting DNA fingerprints produced by Southern blot hybridization are comprised of multiple hypervariable DNA fragments, show somatic and germline stability and are completely specific to an individual. We now show that this technique can be used for forensic purposes; DNA of high relative molecular mass (Mr) can be isolated from 4-yr-old bloodstains and semen stains made on cotton cloth and digested to produce DNA fingerprints suitable for individual identification. Further, sperm nuclei can be separated from vaginal cellular debris, obtained from semen-contaminated vaginal swabs, enabling positive identification of the male donor/suspect. It is envisaged that DNA fingerprinting will revolutionize forensic biology particularly with regard to the identification of rape suspects.     

分子标记技术及其应用

介绍了DNA分子标记的分类以及限制性片段长度多态性、小卫星 (minisatellite)DNA标记、微卫星(microsatellite)DNA标记技术、随机扩增多态性DNA(RAPD)、扩增片段长度多态性 (AFLP)和单核苷酸多态性等主要DNA分子标记的基本原理、优缺点以及它们在人类、动物等研究中的应用概况和展望。     

High mutation rate and predominance of insertions in the Caenorhabditis elegans nuclear genome

Mutations have pivotal functions in the onset of genetic diseases and are the fundamental substrate for evolution. However, present estimates of the spontaneous mutation rate and spectrum are derived from indirect and biased measurements. For instance, mutation rate estimates for Caenorhabditis elegans are extrapolated from observations on a few genetic loci with visible phenotypes and vary over an order of magnitude. Alternative approaches in mammals, relying on phylogenetic comparisons of pseudogene loci and fourfold degenerate codon positions, suffer from uncertainties in the actual number of generations separating the compared species and the inability to exclude biases associated with natural selection. Here we provide a direct and unbiased estimate of the nuclear mutation rate and its molecular spectrum with a set of C. elegans mutation-accumulation lines that reveal a mutation rate about tenfold higher than previous indirect estimates and an excess of insertions over deletions. Because deletions dominate patterns of C. elegans pseudogene variation, our observations indicate that natural selection might be significant in promoting small genome size, and challenge the prevalent assumption that pseudogene divergence accurately reflects the spontaneous mutation spectrum.     

Behaviour modification by in vitro mutagenesis of a variable region within the period gene of Drosophila

The period gene of Drosophila melanogaster, implicated in the control of both the circadian and male courtship song rhythms, is found to be polymorphic. Alleles differ in the length of a region of the gene encoding a series of threonine-glycine repeat units. The phenotypes of transformed fruit flies, in which the only functional period gene lacks the entire perfect threonine-glycine repeat region, show that the effects of the period gene on the circadian and male courtship song rhythms can be dissociated.     

Epistasis and balanced polymorphism influencing complex trait variation

Complex traits such as human disease, growth rate, or crop yield are polygenic, or determined by the contributions from numerous genes in a quantitative manner. Although progress has been made in identifying major quantitative trait loci (QTL), experimental constraints have limited our knowledge of small-effect QTL, which may be responsible for a large proportion of trait variation. Here, we identified and dissected a one-centimorgan chromosome interval in Arabidopsis thaliana without regard to its effect on growth rate, and examined the signature of historical sequence polymorphism among Arabidopsis accessions. We found that the interval contained two growth rate QTL within 210 kilobases. Both QTL showed epistasis; that is, their phenotypic effects depended on the genetic background. This amount of complexity in such a small area suggests a highly polygenic architecture of quantitative variation, much more than previously documented. One QTL was limited to a single gene. The gene in question displayed a nucleotide signature indicative of balancing selection, and its phenotypic effects are reversed depending on genetic background. If this region typifies many complex trait loci, then non-neutral epistatic polymorphism may be an important contributor to genetic variation in complex traits.     

基于金钗石斛EST的短串联重复序列的挖掘

利用计算机程序搜索了金钗石斛EST上的短串联重复位点,共获得2122个基序长度2～7 bp,SIR长度不小于12 bp且重复次数不小于3的SIR位点.其中,3 bp-SIR最为丰富,而2 bp和6 bp基序的STR位点在可表达基因中富集.金钗石斛基因组存在439个基因特异分布的STR位点,暗示这些STR位点可能与特定的...