Analysis of DNA of higher primates using inter-SINE PCR

Two types (MIR and Alu) of short interspersed repeated DNA sequences (SINEs) were used for analysis of genetic relationships among higher primates, and for detection of polymorphism in human genomic DNA. The DNA regions located between the neighboring copies of these SINEs were amplified in polymerase chain reaction with primers complementary to the MIR and Alu consensus sequences (inter-SINE PCR). Comparison of the sets of amplified DNA fragments for different species or individuals provides evaluation of the relationships among them. Using inter-MIR PCR technique, the relationships among the higher primates of the infraorder Catarrhini reported elsewhere were confirmed, pointing to the efficiency of the method for phylogenetic studies. No human DNA polymorphism was revealed with the help of inter-MIR PCR. This polymorphism was detected by means of inter-Alu PCR, which is probably associated with the continuing amplification of Alu elements in human genome.     

DNA PCR typing of non-human primates

Human beings and non human primates show similarities in the non coding DNA range too, but up to now there are only a few data. This paper presents first results of a study dealing with a larger spectrum of species and individuals, considering the genetic marker HLA-DQA1, LDLR, GYPA, HBGG, D7S8, GC (partionally coding) and VWA, FES, F13B, TH01, CD4, FGA (not coding). The results show that not only the apes can be typed but also Macaca sylvanus as a member of the Cercopithecoidea. In contrast to earlier publications there is an unexpected larger similarity between the allele ranges of the apes studied and those of human beings.     

Individual identification of non-human primates using DNA fingerprinting

DNA fingerprints were studied in non-human primates including three species of Old World monkeys and one species of hominoid, using tandem repeats of a 28-base-pair sequence downstream of the human c-Ha-ras-1 oncogene as a probe. We observed Southern hybridization patterns consisting of multiple hypervariable DNA fragments, which were specific to each of the individuals examined. These results indicate that DNA fingerprinting is a powerful tool for identification of individuals among non-human primates, as is the case in man. On leave from the Department of Legal Medicine, Institute of Community Medicine, University of Tsukuba.     

Evolution of DNA on Y-chromosome in hominoid primates as examined by PCR

Every species of non-human primates, especially those of hominoids, has a variety of reproductive structures and accompanying male traits, such as sexual dimorphism and relative size of testis to body weight, which may be at least partly triggered by DNA on the Y-chromosome. Recently, a panel of PCR (Polymerase Chain Reaction) primer sets were designed to amplify various DNA segments spread over the human Y-chromosome. We applied these primer sets for amplification of DNA segments on the Y-chromosome of hominoid species: chimpanzee, bonobo (Pygmy chimpanzee), gorilla, orangutan, whitehanded gibbon, agile gibbon, and Japanese monkey as an out group. The DNA segments including SRY, testis determining factor, and ZFX/ZFY could be amplified clearly in males of all species examined. These highly conserved genes may serve important biological functions. However, as the phylogenic distance from humans increased, some of the DNA segments could not be amplified. For example, DYZ1 (SY160) could be amplified only using human DNA as a template, and DYF60S1 (SY61), DYZ217 (SY126) and DYS233 (SY148) could be amplified only using human and African great ape DNA. It is interesting to note that locus DYS250 (SY17) could not be amplified in chimpanzee and bonobo but amplified in gorilla and orangutan. Locus DYS251 (SY18) was amplified in all species except the white-handed gibbon. These results indicate that a variety of evolutionary events including mutation, deletion, insertion, and rearrangement occurred in Y-chromosome DNA during primate evolution.     

PCR libraries of ancient DNA using a generalized PCR method.

We describe a generalized PCR method that will amplify fragments of DNA without any knowledge of sequence using a single primer. Although we are presently using this method to amplify DNA fragments isolated from ancient preserved tissues, in effect, producing PCR libraries, it may prove to have other applications.     

DNA amplification using PCR with abutting primers

Molecular Biology - DNA analysis of ñîmplex biological objects (wastewater, soil, archaeological and forensic samples, etc.) is currently of great interest. DNA of these objects is...     

降落-重叠PCR法四重融合构建平菇同源重组片段

对多个长片段的基因融合目前仍缺少有效的方法. 本文提出一种新的融合PCR策略,即在常规的重叠PCR的第1步和第2步均增加1个降落PCR程序,减少不适当的退火温度和PCR产物3′端额外碱基A对片段融合、扩增的影响,提高正确融合与扩增的效率. 结果表明,为构建平菇葡聚糖合成酶启动子的同源重组序列,在4个长度分别是1 015 bp、2 822 bp、2 206 bp和1 008 bp的片段进行融合时,在重叠PCR的第1步加上退火温度61.5 ℃～57.5 ℃、每降落0.5 ℃进行1个循环的降落PCR程序,在重叠PCR的第2步加上退火温度60 ℃～56 ℃、每降落0.5 ℃进行1个循环的降落PCR程序,经过1次PCR即获得顺序正确的全长融合片段. 测序结果与4个片段序列的一致性达到98.5%,降落-重叠PCR法对多个长片段的基因 融合具有较高的应用价值.     

De novo DNA synthesis using single molecule PCR

The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.     

Color vision of ancestral organisms of higher primates

The color vision of mammals is controlled by photosensitive proteins called opsins. Most mammals have dichromatic color vision, but hominoids and Old World (OW) monkeys enjoy trichromatic vision, having the blue-, green-, and red-sensitive opsin genes. Most New World (NW) monkeys are either dichromatic or trichromatic, depending on the sex and genotype. Trichromacy in higher primates is believed to have evolved to facilitate the detection of yellow and red fruits against dappled foliage, but the process of evolutionary change from dichromacy to trichromacy is not well understood. Using the parsimony and the newly developed Bayesian methods, we inferred the amino acid sequences of opsins of ancestral organisms of higher primates. The results suggest that the ancestors of OW and NW monkeys lacked the green gene and that the green gene later evolved from the red gene. The fact that the red/green opsin gene has survived the long nocturnal stage of mammalian evolution and that it is under strong purifying selection in organisms that live in dark environments suggests that this gene has another important function in addition to color vision, probably the control of circadian rhythms.      

PCR amplification of streptococcal DNA using crude cell lysates

Gram-positive organisms such as streptococci and enterococci are often difficult to lyse. Obtaining DNA for procedures such as PCR amplification usually requires a large scale isolation for each strain under investigation. We describe a simple procedure for small volumes of whole cells, involving pretreatment with detergent and proteinase that allows for efficient release of DNA for PCR amplification. This procedure is fast, reproducible, can be used with a large number of samples, and has been successfully applied to a variety of streptococcal and enterococcal strains.     

Sexual dimorphisms in dental dimensions of higher primates

Dental dimensions and distributions of dental dimensions of males and females were compared for great apes (Pan, Gorilla, and Pongo, and humans (Homo). The results were examined and discussed with reference to fossil primates Sivapithecus and Ramapithecus. The analyses focused on patterns of sexual dimorphism, both with regard to mean dimensions and the distribution of those dimensions. Sex differences in mean canine dimensions were large and significant for Gorilla and Pongo, significant but smaller for Pan, and small but occasionally significant for Homo. The dispersions of measures were greater for males than for females in Gorilla and Pan but did not differ significantly for Pongo or Homo. Examination of the noncanine teeth revealed complex sex differences. In the anterior teeth, sex differences in mean dimensions were generally apparent for Gorilla and Pongo, less so for Pan, and least of all in Homo. The patterns of dispersion of measures of anterior teeth differed markedly from those of the canines. Pan exhibited the same pattern for anterior and canine teeth. Gorilla showed the opposite pattern. Pongo and Homo showed similar dispersions for males and females in many cases. Sex differences in posterior teeth followed the pattern of the canines for Gorilla and were absent for Pan. Pongo exhibited mean differences in dimensions across sex, but dispersions were similar. The pattern for Homo was most like that of Pongo, but with fewer significant differences. The genera differed with regard to the number of significant differences in means or dispersions along the tooth row. It is clear that the patterns of dimorphism differ qualitatively across all extant genera of great apes and humans. It appears that the pattern for Homo most closely resembles that of Ramapithecus, whereas Pongo most closely resembles Sivapithecus. The patterns for Gorilla and Pan appear to be unlike either of the fossil forms. It is suggested that the qualitatively distinct patterns of dental sexual dimorphism indicate substantial flexibility during recent primate evolution and that the degree of structural flexibility demonstrated provides a basis for appreciating potential for plasticity of gender differences in behavioral, social, and cultural systems.     

MethylScreen: DNA methylation density monitoring using quantitative PCR

Aberrant gene silencing of genes through cytosine methylation has been demonstrated during the development of many types of cancers including prostate cancer Several genes including GSTP1 have been shown to be methylated in prostate cancer leading to the suggestion and demonstration that methylation status of such genes could be used as cancer diagnosis markers alone or in support of histology. We developed a bisulfite-free alternative, MethylScreen technology, an assay for DNA methylation detection utilizing combined restriction from both methylation-sensitive restriction enzymes (MSRE) and methylation-dependent restriction enzymes (MDRE). MethylScreen was used to analyze the 5' region of GSTP1 in cell lines, in vitro methylated DNA populations, and flash-frozen tissue samples in an effort to characterize the output and analytical performance characteristics of the assay. The output from the quantitative PCR assay suggested that it could not only detect fully methylated molecules in a mixed population below the 1% level, but it could also quantify the abundance of intermediately methylated molecules. Interestingly, the interpreted output from the four quantitative PCRs closely resembled the molecular population as described by clone-based bisulfite genomic sequencing.     

Salmonella DNA persistence in natural seawaters using PCR analysis

The risks of false-positive responses were examined when using the polymerase chain reaction (PCR) method for the detection of Salmonella in the marine environment (water and shellfish). The degradation rates of DNA, both free and from dead Salmonella , were evaluated in natural seawaters maintained at 10° and 20°C, using PCR with Vir and invA primers. The DNA of dead Salmonella was detected up to 55 d in seawater collected in winter and stored at 10°C. But in summer, the persistence was shorter: 10 d or even 2 d for a smaller inoculum (3 × 10³ Salmonella ml⁻¹). The role of the planktonic organisms present in spring and summer was pinpointed. For free DNA, the persistence times were shorter: from 2 to 4 d at 20°C, and from 3 to 8 d at 10°C showing that the nuclease activity of marine organisms is higher at warm temperatures. These data led us to recommend careful interpretations of direct PCR results, especially during cold periods and for samples collected close to terrestrial discharges of high concentrations of live, dead or lysed Salmonella . PCR is a rapid, specific and sensitive method, but should be applied with care to marine samples, in order to avoid false-positive responses.     

Molecular evolution of intergenic DNA in higher primates: pattern of DNA changes, molecular clock, and evolution of repetitive sequences

A 3.1-kb intergenic DNA fragment located between the psi beta-globin and delta-globin genes in the beta-globin gene cluster was cloned from gorilla, orangutan, rhesus monkey, and spider monkey, and the nucleotide sequence of each fragment was determined. The phylogeny of these four sequences, together with two previously published allelic sequences from humans and one from chimpanzee, was constructed, and the accumulation of mutations in the region was analyzed. The sites of base substitutions are not evenly distributed within the region: two Alu repeats have accumulated 0.21 + 0.02 substitutions/site with 0.15 + 0.008 substitutions/site in the remainder of the fragment. The occurrence of substitutions at neighboring sites is more frequent than would be expected if they were independent. The observed excesses disappear when ancestral -CG- dinucleotide sites are excluded. The phylogenetic relationships of the sequences indicate that the human sequence shares a most recent coancestor with the chimpanzee sequence. The data also show that great apes have accumulated fewer mutations in this part of the genome than has the rhesus monkey. The relative rates of accumulation of 12 kinds of nucleotide substitution in the region during primate evolution are asymmetric in the DNA strands. From these rates of accumulation, the origin of a simple stretch of sequence near the 3' end of the 3.1-kb fragment was deduced to be a sequence comprising 50% T and 50% C on one strand. The two oppositely oriented Alu sequences in the 3.1-kb region were inserted at their present positions before the divergence of the New-World monkeys from other lineages. Our analysis shows that the nucleotide sequences of the two Alu repeats in spider monkey are unexpectedly similar both to each other and to the deduced ancestral sequence of Alu repeats. The data suggest that there has been some type of recombinational event between the spider monkey Alu repeats but that it was not a simple gene conversion.