Low-stringency PCR Research Articles

Comparing PCR techniques against conventional cercarial shedding methods for detecting Schistosoma mansoni infection in Biomphalaria snails

The detection of Schistosoma mansoni infection in both its intermediate (snail) and definitive (human) hosts is useful in providing information on the transmission of schistosomiasis. Three pairs of previously designed PCR primers (SM1-7, SMF/R & ND5) used for the detection of S. mansoni infection were tested. We assess the utility of each of these primer sets for detecting S. mansoni infection both in artificially exposed laboratory bred Biomphalaria glabrata, and field infected African Biomphalaria sudanica and Biomphalaria pfeifferi. Two of the three primer sets (SMF/R & ND5) detected S. mansoni infection in snails, but amplification of S. mansoni DNA with SM1-7 was unreliable. For the artificially exposed laboratory bred B. glabrata snails, SMF/R and ND5 both detected infection in more snails than the cercarial shedding method. Infection detection rates were 62.4% for ND5, 57.1% for SMF/R and 50.4% using traditional cercarial shedding methods. Both SMF/R and ND5 detected S. mansoni infection in 91% of snails observed shedding cercariae, increasing to 98.5% when low stringency PCR methods were used. When comparing each of the detection methods using a Bayesian latent class analysis model, ND5 had the highest detection sensitivity and negative predictive value (NPV), while SMF/R had the highest detection specificity and positive predictive value (PPV). In field collected Biomphalaria snails, ND5 detected S. mansoni infection in 21 of 24 snails categorised as shedding S. mansoni cercariae and 4 of 24 snails categorised as shedding non-S. mansoni cercariae, while SMF/R detected infection in 18 of 24 snails categorised as shedding S. mansoni cercariae and in 3 of 24 snails categorised as shedding non-S. mansoni cercariae. All SMF/R and ND5 PCR products were shown to be S. mansoni indicating that these field snails must have been infected with both S. mansoni and cercariae from other Schistosoma species. This indicates that the two primer sets are specific for S. mansoni and will not amplify non-S. mansoni species when used at their recommended annealing temperatures. Both the SMF/R and ND5 primers effectively detected S. mansoni infection in three Biomphalaria species and have improved detection sensitivity over cercarial shedding.

DDRT-PCR approaches applied for preeminent results in the isolation of DETs from fish brain tissues.

Differential Display (DD) is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored. Thus, the present work aimed to describe DD technique modifications in order to improve outcomes related to the isolation of DETs (Differentially Expressed Transcripts), using Leporinus macrocephalus, a large commercially exploited South American species, as a fish design. Different DDRT-PCR approaches were applied to brain samples and the products of the reactions were analyzed on 6% polyacrylamide gels stained with 0.17% Silver Nitrate (AgNO3). The use of PCR reactions under high stringency conditions and longer oligonucleotides based on VNTR (Variable Number of Tandem Repeats) core sequences led to better results when compared to low stringency PCR conditions and the use of decamer oligonucleotides. The improved approach led to the isolation of differentially expressed transcripts on adult males and females of L. macrocephalus. This study indicates that some modifications on the DDRT-PCR method can ensure isolation of DETs from different fish tissues and the development of robust data related to this approach.

Human papillomavirus type 16 viral load measurement as a predictor of infection clearance

Viral load measurements may predict whether human papillomavirus (HPV) type 16 infections may become persistent and eventually lead to cervical lesions. Today, multiple PCR methods exist to estimate viral load. We tested three protocols to investigate viral load as a predictor of HPV clearance. We measured viral load in 418 HPV16-positive cervical smears from 224 women participating in the Ludwig-McGill Cohort Study by low-stringency PCR (LS-PCR) using consensus L1 primers targeting over 40 known HPV types, and quantitative real-time PCR (qRT-PCR) targeting the HPV16 E6 and L1 genes. HPV16 clearance was determined by MY09/11 and PGMY PCR testing on repeated smears collected over 5 years. Correlation between viral load measurements by qRT-PCR (E6 versus L1) was excellent (Spearman's rank correlation, ρ = 0.88), but decreased for L1 qRT-PCR versus LS-PCR (ρ = 0.61). Viral load by LS-PCR was higher for HPV16 and related types independently of other concurrent HPV infections. Median duration of infection was longer for smears with high copy number by all three PCR protocols (log rank P<0.05). Viral load is inversely related to HPV16 clearance independently of concurrent HPV infections and PCR protocol.

Cloning of anthozoan fluorescent protein genes

Many cnidarians display vivid fluorescence under proper lighting conditions. In general, these colors are due to the presence of fluorescent proteins similar to the green fluorescent protein (GFP) originally isolated from the hydrozoan medusa Aequorea victoria (Cnidaria: Hydrozoa). To optimize the search for new fluorescent proteins (FPs), a technique was developed that allows for the rapid cloning and screening of FP genes without the need for a prior knowledge of gene sequence. Using this method, four new FP genes were cloned, a green from Montastraea cavernosa (Anthozoa: Scleractinia: Faviidae), a cyan from Pocillopora damicornis (Anthozoa: Scleractinia: Pocilloporidae), a cyan from Discosoma striata (Anthozoa: Corallimorpharia), and a red from a second Discosoma species. Two additional green FPs were cloned, one from M. cavernosa and one from its congener Montastraea faveolata, from purified cDNA using PCR primers designed for the first M. cavernosa green FP. Each FP has recognizable amino acid sequence motifs that place them conclusively in the GFP protein family. Mutation of these products using a low-stringency PCR protocol followed by screening of large numbers of bacterial colonies allowed rapid creation of mutants with a variety of characteristics, including changes in color, maturation time, and brightness. An enhanced version of the new red FP, DspR1+, matures faster at 30 °C than the commercially available DsRed but matures slower than DsRed at 37 °C. One of the M. cavernosa green FPs, McaG2, is highly resistant to photobleaching and has a fluorescence quantum yield approximately twice that of EGFP-1.

Association of human papillomavirus viral load with HPV16 and high-grade intraepithelial lesion

The aim of the present study was to evaluate the relationship between viral type and copy number of human papillomavirus (HPV) with respect to the grade of cervical disease, and also to identify the existence of an HPV type-dependent viral load effect. DNA from 275 exocervical specimens, previously evaluated for histologic diagnosis, were evaluated for HPV presence, HPV type, and viral load. Viral load determination was performed using the low stringency PCR method (LS-PCR). Significant differences were found between the samples infected with HPV16 with respect to the samples infected with other ‘high-risk’ viral types (HPV −18, −31, −33 or −51) and ‘low-risk’ types (P &lt; 0.05). However, highly significant differences were found between the viral loads observed in the high-grade squamous intraepithelial lesions group and normal epithelium (OR = 8.53) or the low grade ones (OR = 3.10). Moreover, a high viral load was detected in the condyloma acuminatum group compared to the normal epithelia samples (p&lt; 0.05). This work confirms the genotype-specific association of viral load to the presence of HPV16. Also, a trend to higher viral loads could be seen in the more compromised cervical lesions. An unexpected level of viral particles appeared associated to the condylomas. This fact could be explained by a productive infection with high levels of viral replication.

Association of human papillomavirus viral load with HPV16 and high-grade intraepithelial lesion.

The aim of the present study was to evaluate the relationship between viral type and copy number of human papillomavirus (HPV) with respect to the grade of cervical disease, and also to identify the existence of an HPV type-dependent viral load effect. DNA from 275 exocervical specimens, previously evaluated for histologic diagnosis, were evaluated for HPV presence, HPV type, and viral load. Viral load determination was performed using the low stringency PCR method (LS-PCR). Significant differences were found between the samples infected with HPV16 with respect to the samples infected with other 'high-risk' viral types (HPV -18, -31, -33 or -51) and 'low-risk' types (P < 0.05). However, highly significant differences were found between the viral loads observed in the high-grade squamous intraepithelial lesions group and normal epithelium (OR = 8.53) or the low grade ones (OR = 3.10). Moreover, a high viral load was detected in the condyloma acuminatum group compared to the normal epithelia samples (p< 0.05). This work confirms the genotype-specific association of viral load to the presence of HPV16. Also, a trend to higher viral loads could be seen in the more compromised cervical lesions. An unexpected level of viral particles appeared associated to the condylomas. This fact could be explained by a productive infection with high levels of viral replication.

Development of a quantitative competitive reverse transcriptase polymerase chain reaction for the quantification of growth hormone gene expression in pigs

After the advent of the genome projects, followed by the discovery of DNA polymorphisms, basic understanding of gene expression is the next focus to explain the association between polymorphisms and the level of gene expression, as well as to demonstrate the interaction among genes. Among the various techniques for the investigation of transcriptional profiling involving patterns of gene expression, quantitative PCR is the simplest analytical laboratory technique. The objective of this work was to analyze two strategies of a competitive PCR technique for the quantification of the pig growth hormone (GH) gene expression. A pair of primers was designed targeting exons 3 and 5, and two competitive PCR strategies were performed, one utilizing a specific amplicon as a competitor, and the other utilizing a low-stringency PCR amplicon as a competitor. The latter strategy proved to be easier and more efficient, offering an accessible tool that can be used in any kind of competitive reaction, facilitating the study of gene expression patterns for both genetics and diagnostics of infectious diseases.

Viral load as a predictor of the risk of cervical intraepithelial neoplasia.

HPV infections are believed to be a necessary cause of cervical cancer. Viral burden, as a surrogate indicator for persistence, may help predict risk of subsequent SIL. We used results of HPV test and cytology data repeated every 4-6 months in 2,081 women participating in a longitudinal study of the natural history of HPV infection and cervical neoplasia in São Paulo, Brazil. Using the MY09/11 PCR protocol, 473 women were positive for HPV DNA during the first 2 visits. We retested all positive specimens by a quantitative, low-stringency PCR method to measure viral burden in cervical cells. Mean viral loads and 95% CIs were calculated using log-transformed data. RRs and 95% CIs of incident SIL were calculated by proportional hazards models, adjusting for age and HPV oncogenicity. The risk of incident lesions increased with viral load at enrollment. The mean number of viral copies/cell at enrollment was 2.6 for women with no incident lesions and increased (trend p = 0.003) to 15.1 for women developing 3 or more SIL events over 6 years of follow-up. Compared to those with <1 copy per cell in specimens tested during the first 2 visits, RRs for incident SIL increased from 1.9 (95% CI 0.8-4.2) for those with 1-10 copies/cell to 4.5 (95% CI 1.9-10.7) for those with >1,000 copies/cell. The equivalent RR of HSIL for >1,000 copies/cell was 2.6 (95% CI 0.5-13.2). Viral burden appears to have an independent effect on SIL incidence. Measurement of viral load, as a surrogate for HPV persistence, may identify women at risk of developing cervical cancer precursors.

Diversity of 16S rDNA and naphthalene dioxygenase genes from coal-tar-waste-contaminated aquifer waters.

Microbial diversity in four wells along a groundwater flowpath in a coal-tar-waste-contaminated aquifer was examined using RFLP analysis of both 16S rDNA and naphthalene dioxygenase (NDO) genes. Amplified ribosomal DNA restriction analysis (ARDRA) relied upon eubacteria-specific primers to generate four clone libraries. From each library, 100 clones were randomly picked for analysis. Sixty percent of 400 clones contained unique ARDRA patterns. Diversity indices calculated for each community were high (Shannon-Weaver, H = 3.53 to 3.69). Clones representing ARDRA patterns found in the highest abundance were sequenced (31 total). Sequences related to aerobic bacteria (e.g., Nitrospira, Methylomonas, and Gallionella) predominated among those retrieved from the uncontaminated area of the site, whereas sequences related to facultatively aerobic and anaerobic bacteria (e.g. Azoarcus, Syntrophus, and Desulfotomaculum) predominated among those retrieved from contaminated areas of the site. Using NDO-specific primers and low-stringency PCR conditions, variability in RFLP patterns was only detected in community-derived DNA (3 of 4 wells) and not in 5 newly isolated naphthalene-degrading pure cultures. The ARDRA patterns of the pure culture isolates were not found in the clone libraries. Polymorphisms in community 16S rDNA and NDO genes found in well-water microorganisms reflected distinctive geochemical conditions across the site. Sequences related to sulfate-reducing bacteria were found in groundwater that contained sulfide, while sequences related to Gallionella, Syntrophus, and nitrate-reducing aromatic hydrocarbon-degrading bacteria were found in groundwater that contained ferrous iron, methane, and naphthalene, respectively.

Molecular evolution of calmodulin and calmodulin-like genes in the cephalochordate Branchiostoma.

Calmodulin is a calcium-binding EF-hand protein that is an activator of many enzymes as well as ion pumps and channels. Due to its multiple targets and its central role in the cell, understanding the evolutionary history of calmodulin genes should provide insights into the origin of genetic complexity in eukaryotes. We have previously isolated and characterized a calmodulin gene from the early-diverging chordate Branchiostoma lanceolatum (CaM1). In this paper, we report the existence of a second calmodulin gene (CaM2) as well as two CaM-like genomic fragments (CaML-2, CaML-3) in B. lanceolatum and a CaM2 and three CaM-like genes (CaML-1, CaML-2, CaML-3) in B. floridae. The CaM-like genes were isolated using low-stringency PCR. Surprisingly, the nucleotide sequences of the B. lanceolatum CaM1 and CaM2 cDNAs differ by 19.3%. Moreover, the CaM2 protein differs at two positions from the amino acid sequence of CaM1; the latter is identical to calmodulins in Drosophila melanogaster, the mollusc Aplysia californica, and the tunicate Halocynthia roretzi. The two B. lanceolatum CaM-like genes are more closely related to the CaM2 than to the CaM1 gene. This relationship is supported by the phylogenetic analyses and the identical exon/intron organization of these three genes, a relationship unique among animal CaM sequences. These data demonstrate the existence of a CaM multigene family in the cephalochordate Branchiostoma, which may have evolved independently from the multigene family in vertebrates.

Mapping of intra-locus duplications and introgressed DNA: aids to map-based cloning of genes from complex genomes illustrated by physical analysis of the Rx locus in tetraploid potato

We describe here novel approaches to high-resolution mapping of repeated and introgressed DNA which may prove generally useful in map-based cloning from complex genomes. These approaches were developed in order to clone the Rx locus in potato. First, we prepared a BAC library from a tetraploid plant carrying Rx in the duplex condition (Rx, Rx, rx, rx). BAC clones were then isolated with close markers on either side of Rx. However these clones did not extend across Rx: in the cloned DNA the closest markers to Rx were separated by single recombination events on either side of Rx. To bridge the gap we exploited the finding that the BAC clones on the right side of Rx contained resistance-gene homologues. Anticipating that there would be duplicated copies of these resistance gene homologues in the vicinity of Rx, we used low-stringency PCR conditions to identify additional markers. One of these markers was completely linked to Rx in our mapping population and was used to isolate a BAC (BAC77) that had not been previously identified by screening with Rx-flanking markers. Based on two criteria it was concluded that BAC77 spans Rx. There was a chromosomal recombination in one plant of our mapping population that separated the BAC77 right end from Rx. On the other side of Rx it was found that the BAC77 left end was outside the region of DNA carrying Rx that had been introgressed into potato cv Amaryl from a Solanum tuberosum ssp. andigena accession CPC1673.

Identification and complete cDNA sequence of the missing Drosophila MCMs: DmMCM3, DmMCM6 and DmMCM7.

The minichromosome maintenance (MCM) gene family consists of six members (MCM2, 3, 4, 5, 6 and 7) in Saccharomyces cerevisiae as well as in humans. Each family member plays an essential role in the replication of DNA. In Drosophila melanogaster only three members, DmMCM2, DmMCM4/dpa and DmMCM5/DmCDC46, have been studied. In addition, two other partial sequences were recently reported. Using degenerate primers and low stringency PCR conditions six different DNA sequences were identified with highest sequence similarity to MCM2, 3, 4, 5, 6 and 7. Sequence analysis of full length cDNA clones corresponding to the MCM3, 6 and 7 fragment proves the existence of six MCM genes in Drosophila melanogaster. Strong homology to the human counterparts, mRNA expression analysis and physico-chemical properties suggest a conserved function in DNA replication for DmMCM3, 6 and 7.

BMPR-II expression and OP-1 effects in developing chicken retinal explants.

Low-stringency PCR was used to isolate the chicken homologue of the human bone morphogenetic protein receptor type II (BMPR-II). In situ hybridization localized mRNA expression for this serine/threonine kinase receptor. It was weakly expressed in the vitreal side of the neural retina at E6. In the E7 chicken, fairly strong labelling was found in cells of the internal part of the neural retina. At E9, strong labelling was found in the region of the retinal ganglion cells. Explants of E6 retina exposed to osteogenic protein-1 (OP-1/BMP-7), exhibited dense retinal fibre outgrowth. This suggests that BMPR-II may form a signalling receptor complex important for retinal development. OP-1 and related ligands may serve functions supplementary to those of neurotrophins.

Genetic variability and identification of the intermediate snail hosts of Schistosoma mansoni.

Studies based on shell or reproductive organ morphology and genetic considerations suggest extensive intraspecific variation in Biomphalaria snails. The high variability at the morphological and genetic levels, as well as the small size of some specimens and similarities between species complicate the correct identification of these snails. Here we review our work using methods based on polymerase chain reaction (PCR) amplification for analysis of genetic variation and identification of Biomphalaria snails from Brazil, Argentina, Uruguay and Paraguay. Arbitrarily primed-PCR revealed that the genome of B. glabrata exhibits a remarkable degree of intraspecific polymorphism. Low stringency-PCR using primers for 18S rRNA permitted the identification of B. glabrata, B. tenagophila and B. occidentalis. The study of individuals obtained from geographically distinct populations exhibits significant intraspecific DNA polymorphism, however, specimens from the same species, exhibit some species specific LSPs. We also showed that PCR-restriction fragment of length polymorphism of the internal transcribed spacer region of Biomphalaria rDNA, using Ddel permits the differentiation of the three intermediate hosts of Schistosoma mansoni. the molecular biological techniques used in our studies are very useful for the generation of new knowledge concerning the systematics and population genetics of Biomphalaria snails.

The Eek receptor, a member of the Eph family of tyrosine protein kinases, can be activated by three different Eph family ligands.

The Eph family of receptors, the largest subgroup within the tyrosine protein kinase receptor family, are comprised of at least thirteen members, many of which are predominantly expressed in the developing and adult nervous system. In this study, we have isolated a full-length cDNA, encoding the mouse homologue of a previous partially characterized Eek protein, a member of Eph receptor tyrosine kinase family. In a comparison of the amino acid sequences of various Eph family members, Eek is most similar to Ehk-3/MDK1, Sek/Cek8, Ehk-2, Hek/Mek4/Cek4, and Bsk/Ehk1/Rek7/Cek7, which are predominantly expressed in the nervous system. Additionally, we have used a low-stringency PCR cloning technique to identify ligands, related to B61, that may interact with Eek. Three different GPI-linked ligands, namely Elf-1/Cek7-L, Ehk1-L/Efl-2/Lerk3 and AL-1/RAGS, were isolated from mouse brain. To study the functional interactions between these ligands and the Eek receptors, we have constructed chimeric ligands consisting of the Fc portion of human IgG fused to their carboxyl-terminus. These chimeric ligands bound to, and activated both the Eek receptors and the Eek-TrkB chimeric receptors expressed in NIH3T3 cells. These findings suggest that Eek receptor can be activated by at least three different GPI-linked ligands.

Differentiation of Leptospira species and serovars by PCR-restriction endonuclease analysis, arbitrarily primed PCR and low-stringency PCR.

Reference strains from 30 serovars representing seven species of Leptospira and 48 recent isolates from human patients, dogs and rats, were characterised by polymerase chain reaction-restriction endonuclease analysis (PCR-REA), arbitrarily primed PCR (AP-PCR) and low stringency PCR (LS-PCR). PCR-REA analysis yielded seven groups among 29 serovars of pathogenic Leptospira; the non-pathogenic L. biflexa serovar patoc was not amplified with the primer pairs studied. AP-PCR and LS-PCR fingerprinting resulted in 25 and 21 distinct profiles, respectively, among the 30 reference strains. The results of the three PCR-based techniques were highly concordant and were in general agreement with those from previous DNA studies, confirming the high level of polymorphism among Leptospira species and serovars, and supported the concept of the serovar as the basic taxonomic unit of leptospiral classification. Results of the PCR-based typing methods for 11 randomised leptospiral strains, 36 clinical isolates from human patients and dogs and 12 survey isolates from trapped rats agreed with those from serological identification. With one exception, isolates of the same serovar gave identical profiles irrespective of the source. AP-PCR and LS-PCR are simple to perform and interpret, and appear to be useful for characterising isolates of Leptospira spp. for diagnostic and epidemiological purposes.

Genotype-related fingerprints from HLA-DPB1 exon 2 low-stringency PCR.

Techniques based on the formation of heteroduplexes between relevant heterologous amplified HLA loci have proved to be simple and cost-effective screening methods for the detection of DNA sequence diversity. However, the banding patterns produced may not be as complex as required. We used the original procedure of Pena et al. (Proceedings of the National Academy of Sciences USA 91, 1946-1949, 1994) to generate fingerprints from a specific, polymorphic PCR product. HLA-DPB1 exon 2 was amplified, recovered from agarose gel, and used as a template for subsequent low-stringency (30 degrees C) amplification cycles (LS-PCR) in the presence of a single primer. The LS-PCR products were run on 8% PAGE and silver-stained. In total, 22 subjects were characterized by this method. The issues of the reproducibility and specificity of the patterns obtained were addressed by comparing fingerprints from individuals with the same genotype. The results showed that LS-PCR was robust. A further step was the evaluation of the diversity that can be generated, i.e. the sensitivity of the method. Genotype-related fingerprints were produced, and differences as small as a single nucleotide in heterozygous samples could be detected. We then demonstrated the usefulness of LS-PCR in the evaluation of donor/recipient pairs. We believe that LS-PCR may be a valuable adjunct to the battery of tests aimed at the verification of HLA matching before unrelated bone marrow transplantation. We suggest that it could be used to speed up the search process when several candidate donors are retrieved from registries before embarking on SSOP typing or sequencing.

Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase.

A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed.

Selection of specific gene probes by combined use of low-stringency PCR amplification and Southern-blot hybridization.

We have developed a protocol to isolate a gene from which only limited (amino-acid) sequence information is available. It involves two PCR amplifications using one constant primer and a set of nested primers and subsequent crosswise Southern hybridization. The amplified DNA giving a signal in both lanes is further processed for use in gene bank screening by applying standard procedures. In this way the structural gene for a thiol protease, BLH1, the homologue of the bleomycin A (a cancerostatic drug) resistance gene of rabbit (and man), was isolated from yeast genomic DNA.

Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking

Isolation of DNA segments adjacent to known sequences is a tedious task in genome-related research. We have developed an efficient PCR strategy that overcomes the shortcomings of existing methods and can be automated. This strategy, thermal asymmetric in-ter laced (TAIL)-PCR, utilizes nested sequence-specific primers together with a shorter arbitrary degenerate primer so that the relative amplification efficiencies of specific and nonspecific products can be thermally controlled. One low-stringency PCR cycle is carried out to create annealing site(s) adapted for the arbitrary primer within the unknown target sequence bordering the known segment. This sequence is then preferentially and geometrically amplified over nontarget ones by interspersion of high-stringency PCR cycles with reduced-stringency PCR cycles. We have exploited the efficiency of this method to expedite amplification and sequencing of insert end segments from P1 and YAC clones for chromosome walking. In this study we present protocols that are amenable to automation of amplification and sequencing of insert end sequences directly from cells of P1 and YAC clones.