Steve Rozen

Primer3 on the WWW for general users and for biologist programmers.

1. Introduction Designing PCR and sequencing primers are essential activities for molecular biologists around the world. This chapter assumes acquaintance with the principles and practice of PCR, as outlined in, for example, refs. 1–4. Primer3 is a computer program that suggests PCR primers for a variety of applications, for example to create STSs (sequence tagged sites) for radiation hybrid mapping (5), or to amplify sequences for single nucleotide polymor-phism discovery (6). Primer3 can also select single primers for sequencing reactions and can design oligonucleotide hybridization probes. In selecting oligos for primers or hybridization probes, Primer3 can consider many factors. These include oligo melting temperature, length, GC content , 3′ stability, estimated secondary structure, the likelihood of annealing to or amplifying undesirable sequences (for example interspersed repeats), the likelihood of primer–dimer formation between two copies of the same primer, and the accuracy of the source sequence. In the design of primer pairs Primer3 can consider product size and melting temperature, the likelihood of primer– dimer formation between the two primers in the pair, the difference between primer melting temperatures, and primer location relative to particular regions of interest or to be avoided.

The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes

The male-specific region of the Y chromosome, the MSY, differentiates the sexes and comprises 95% of the chromosomes length. Here, we report that the MSY is a mosaic of heterochromatic sequences and three classes of euchromatic sequences: X-transposed, X-degenerate and ampliconic. These classes contain all 156 known transcription units, which include 78 protein-coding genes that collectively encode 27 distinct proteins. The X-transposed sequences exhibit 99% identity to the X chromosome. The X-degenerate sequences are remnants of ancient autosomes from which the modern X and Y chromosomes evolved. The ampliconic class includes large regions (about 30% of the MSY euchromatin) where sequence pairs show greater than 99.9% identity, which is maintained by frequent gene conversion (non-reciprocal transfer). The most prominent features here are eight massive palindromes, at least six of which contain testis genes.

Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction

BackgroundChoosing appropriate primers is probably the single most important factor affecting the polymerase chain reaction (PCR). Specific amplification of the intended target requires that primers do not have matches to other targets in certain orientations and within certain distances that allow undesired amplification. The process of designing specific primers typically involves two stages. First, the primers flanking regions of interest are generated either manually or using software tools; then they are searched against an appropriate nucleotide sequence database using tools such as BLAST to examine the potential targets. However, the latter is not an easy process as one needs to examine many details between primers and targets, such as the number and the positions of matched bases, the primer orientations and distance between forward and reverse primers. The complexity of such analysis usually makes this a time-consuming and very difficult task for users, especially when the primers have a large number of hits. Furthermore, although the BLAST program has been widely used for primer target detection, it is in fact not an ideal tool for this purpose as BLAST is a local alignment algorithm and does not necessarily return complete match information over the entire primer range.ResultsWe present a new software tool called Primer-BLAST to alleviate the difficulty in designing target-specific primers. This tool combines BLAST with a global alignment algorithm to ensure a full primer-target alignment and is sensitive enough to detect targets that have a significant number of mismatches to primers. Primer-BLAST allows users to design new target-specific primers in one step as well as to check the specificity of pre-existing primers. Primer-BLAST also supports placing primers based on exon/intron locations and excluding single nucleotide polymorphism (SNP) sites in primers.ConclusionsWe describe a robust and fully implemented general purpose primer design tool that designs target-specific PCR primers. Primer-BLAST offers flexible options to adjust the specificity threshold and other primer properties. This tool is publicly available at http://www.ncbi.nlm.nih.gov/tools/primer-blast.

Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA–binding protein gene

We have detected deletions of portions of the Y chromosome long arm in 12 of 89 men with azoospermia (no sperm in semen). No Y deletions were detected in their male relatives or in 90 other fertile males. The 12 deletions overlap, defining a region likely to contain one or more genes required for spermatogenesis (the Azoospermia Factor, AZF). Deletion of the AZF region is associated with highly variable testicular defects, ranging from complete absence of germ cells to spermatogenic arrest with occasional production of condensed spermatids. We find no evidence of YRRM genes, recently proposed as AZF candidates, in the AZF region. The region contains a single–copy gene, DAZ (Deleted in AZoospermia), which is transcribed in the adult testis and appears to encode an RNA binding protein. The possibility that DAZ is AZF should now be explored.

An STS-Based Map of the Human Genome

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men

Deletions of the AZFc (azoospermia factor c) region of the Y chromosome are the most common known cause of spermatogenic failure. We determined the complete nucleotide sequence of AZFc by identifying and distinguishing between near-identical amplicons (massive repeat units) using an iterative mapping–sequencing process. A complex of three palindromes, the largest spanning 3 Mb with 99.97% identity between its arms, encompasses the AZFc region. The palindromes are constructed from six distinct families of amplicons, with unit lengths of 115–678 kb, and may have resulted from tandem duplication and inversion during primate evolution. The palindromic complex contains 11 families of transcription units, all expressed in testis. Deletions of AZFc that cause infertility are remarkably uniform, spanning a 3.5-Mb segment and bounded by 229-kb direct repeats that probably served as substrates for homologous recombination.

Abundant gene conversion between arms of palindromes in human and ape Y chromosomes

Eight palindromes comprise one-quarter of the euchromatic DNA of the male-specific region of the human Y chromosome, the MSY. They contain many testis-specific genes and typically exhibit 99.97% intra-palindromic (arm-to-arm) sequence identity. This high degree of identity could be interpreted as evidence that the palindromes arose through duplication events that occurred about 100,000 years ago. Using comparative sequencing in great apes, we demonstrate here that at least six of these MSY palindromes predate the divergence of the human and chimpanzee lineages, which occurred about 5 million years ago. The arms of these palindromes must have subsequently engaged in gene conversion, driving the paired arms to evolve in concert. Indeed, analysis of MSY palindrome sequence variation in existing human populations provides evidence of recurrent arm-to-arm gene conversion in our species. We conclude that during recent evolution, an average of approximately 600 nucleotides per newborn male have undergone Y–Y gene conversion, which has had an important role in the evolution of multi-copy testis gene families in the MSY.

The DAZ gene cluster on the human Y chromosome arose from an autosomal gene that was transposed, repeatedly amplified and pruned

It is widely believed that most or all Y–chromosomal genes were once shared with the X chromosome. The DAZ gene is a candidate for the human Y–chromosomal Azoospermia Factor (AZF). We report multiple copies of DAZ (>99% identical in DNA sequence) clustered in the AZF region and a functional DAZ homologue (DAZH) on human chromosome 3. The entire gene family appears to be expressed in germ cells. Sequence analysis indicates that the Y–chromosomal DAZ cluster arose during primate evolution by (i) transposing the autosomal gene to the Y, (ii) amplifying and pruning exons within the transposed gene and (iii) amplifying the modified gene. These results challenge prevailing views of sex chromosome evolution, suggesting that acquisition of autosomal fertility genes is an important process in Y chromosome evolution.

Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection

Many human Y-chromosomal deletions are thought to severely impair reproductive fitness, which precludes their transmission to the next generation and thus ensures their rarity in the population. Here we report a 1.6-Mb deletion that persists over generations and is sufficiently common to be considered a polymorphism. We hypothesized that this deletion might affect spermatogenesis because it removes almost half of the Y chromosomes AZFc region, a gene-rich segment that is critical for sperm production. An association study established that this deletion, called gr/gr, is a significant risk factor for spermatogenic failure. The gr/gr deletion has far lower penetrance with respect to spermatogenic failure than previously characterized Y-chromosomal deletions; it is often transmitted from father to son. By studying the distribution of gr/gr-deleted chromosomes across the branches of the Y chromosomes genealogical tree, we determined that this deletion arose independently at least 14 times in human history. We suggest that the existence of this deletion as a polymorphism reflects a balance between haploid selection, which culls gr/gr-deleted Y chromosomes from the population, and homologous recombination, which continues to generate new gr/gr deletions.

A comprehensive survey of genomic alterations in gastric cancer reveals systematic patterns of molecular exclusivity and co-occurrence among distinct therapeutic targets

Objective Gastric cancer is a major gastrointestinal malignancy for which targeted therapies are emerging as treatment options. This study sought to identify the most prevalent molecular targets in gastric cancer and to elucidate systematic patterns of exclusivity and co-occurrence among these targets, through comprehensive genomic analysis of a large panel of gastric cancers. Design Using high-resolution single nucleotide polymorphism arrays, copy number alterations were profiled in a panel of 233 gastric cancers (193 primary tumours, 40 cell lines) and 98 primary matched gastric non-malignant samples. For selected alterations, their impact on gene expression and clinical outcome were evaluated. Results 22 recurrent focal alterations (13 amplifications and nine deletions) were identified. These included both known targets (FGFR2, ERBB2) and also novel genes in gastric cancer (KLF5, GATA6). Receptor tyrosine kinase (RTK)/RAS alterations were found to be frequent in gastric cancer. This study also demonstrates, for the first time, that these alterations occur in a mutually exclusive fashion, with KRAS gene amplifications highlighting a clinically relevant but previously underappreciated gastric cancer subgroup. FGFR2-amplified gastric cancers were also shown to be sensitive to dovitinib, an orally bioavailable FGFR/VEGFR targeting agent, potentially representing a subtype-specific therapy for FGFR2-amplified gastric cancers. Conclusion The study demonstrates the existence of five distinct gastric cancer patient subgroups, defined by the signature genomic alterations FGFR2 (9% of tumours), KRAS (9%), EGFR (8%), ERBB2 (7%) and MET (4%). Collectively, these subgroups suggest that at least 37% of gastric cancer patients may be potentially treatable by RTK/RAS directed therapies.