Discriminant analysis and its application in DNA sequence motif recognition

Identification of functional motifs in a DNA sequence is fundamentally a statistical pattern recognition problem. Discriminant analysis is widely used for solving such problems. This paper will review two basic parametric methods: LDA (linear discriminant analysis) and QDA (quadratic discriminant analysis). Their usage in recognition of splice sites and exons in the human genome will be demonstrated.     

Pattern recognition in genetic sequences by mismatch density

A new development is introduced here in the use of dynamic programming in finding pattern similarities in genetic sequences, as was first done by Needleman and Wunsch (1969). A condition of pattern similarity is defined and an algorithm is given which scans any set of similarities and screens out those which fail to meet the condition. When the set to be scanned contains every pair of segments, one from each of two given sequences of lengthsm andn (i.e. every possible location for a pattern similarity), then it completes the scan in a number of computational steps proportional tom·n, leaving those pairs of segments which satisfy the similarity condition. The algorithm is based on the concept of match density, as suggested by Goad and Kanehisa (1982).     

Headloop suppression PCR and its application to selective amplification of methylated DNA sequences

Selective amplification in PCR is principally determined by the sequence of the primers and the temperature of the annealing step. We have developed a new PCR technique for distinguishing related sequences in which additional selectivity is dependent on sequences within the amplicon. A 5′ extension is included in one (or both) primer(s) that corresponds to sequences within one of the related amplicons. After copying and incorporation into the PCR product this sequence is then able to loop back, anneal to the internal sequences and prime to form a hairpin structure—this structure is then refractory to further amplification. Thus, amplification of sequences containing a perfect match to the 5′ extension is suppressed while amplification of sequences containing mismatches or lacking the sequence is unaffected. We have applied Headloop PCR to DNA that had been bisulphite-treated for the selective amplification of methylated sequences of the human GSTP1 gene in the presence of up to a 10⁵-fold excess of unmethylated sequences. Headloop PCR has a potential for clinical application in the detection of differently methylated DNAs following bisulphite treatment as well as for selective amplification of sequence variants or mutants in the presence of an excess of closely related DNA sequences.     

DNA-binding activity of the A-factor receptor protein and its recognition DNA sequences

The A-factor receptor protein (ArpA) containing an α-helix-turn-α-helix DNA-binding consensus sequence at its N-terminal portion plays a key role in the regulation of secondary metabolism and cell differentiation in Streptomyces griseus . A binding site forming a palindrome 24 bp in length was initially recovered from a pool of random-sequence oligonucleotides by rounds of a binding/immunoprecipitation/amplification procedure with histidine-tagged ArpA and anti-ArpA antibody. By means of further binding/gel retardation/amplification experiments on the basis of the recovered sequence, a 22 bp palindromic binding site with the sequence 5'-GG(T/C)CGGT(A/T)(T/C)-G(T/G)-3' as one half of the palindrome was deduced as a consensus sequence recognized and bound by ArpA. ArpA did not bind to the binding site in the presence of its ligand, A-factor. In addition, exogenous addition of A-factor to the ArpA–DNA complex induced immediate release of ArpA from the DNA. All of these data are consistent with the idea, obtained from previous genetic studies, that ArpA acts as a repressor-type regulator for secondary metabolism and cellular differentiation by preventing the expression of a certain key gene(s) during the early growth phase. A-factor, produced in a growth-dependent manner, releases ArpA from the DNA, thus switching on the expression of the key gene(s), leading to the onset of secondary metabolism and aerial mycelium formation.     

Identification of consensus patterns in unaligned DNA sequences known to be functionally related

We have developed a method for identifying consensus patternsin a set of unaligned DNA sequences known to bind a common proteinor to have some other common biochemical function. The methodis based on a tnatrix representation of binding site patterns.Each row of the matrix represents one of the four possible bases,each column represents one of the positions of the binding siteand each element is determined by the frequency the indicatedbase occurs at the indicated position. The goal of the methodis to find the most significant matrix-i.e. the one with thelowest probability of occurring by chance-out of all the matricesthat can be formed from the set of related sequences. The reliabilityof the method improves with the number of sequences, while thetime required increases only linearly with the number of sequences.To test this method, we analysed 11 DNA sequences containingpromoters regulated by the Escherichia coli LexA protein. Thematrices we' found were consistent with the known consensussequence, and could distinguish the generally accepted LexAbinding sites from other DNA sequences. Received on November 6, 1989; accepted on December 20, 1989     

pBR322 contains glucocorticoid regulatory element DNA consensus sequences

A computer search of the pBR322 DNA sequence identified five sites matching reported glucocorticoid regulatory element (GRE) DNA consensus sequences and three related sites. A pBR322 DNA fragment containing one GRE site was shown to bind immobilized HeLa S3 cell glucocorticoid receptor and to compete for receptor binding in a competitive binding assay. Conversely, a pBR322 DNA fragment devoid of GRE sites showed barely detectable interaction with glucocorticoid receptor in either of these assays. These results demonstrate the importance of GRE consensus sequences in glucocorticoid receptor interactions with DNA, and further identify a cause for high background binding observed when pBR322 DNA is used as a negative control in studies of glucocorticoid receptor-DNA interactions.     

OrgConv: detection of gene conversion using consensus sequences and its application in plant mitochondrial and chloroplast homologs

Background  

The ancestry of mitochondria and chloroplasts traces back to separate endosymbioses of once free-living bacteria. The highly reduced genomes of these two organelles therefore contain very distant homologs that only recently have been shown to recombine inside the mitochondrial genome. Detection of gene conversion between mitochondrial and chloroplast homologs was previously impossible due to the lack of suitable computer programs. Recently, I developed a novel method and have, for the first time, discovered recurrent gene conversion between chloroplast mitochondrial genes. The method will further our understanding of plant organellar genome evolution and help identify and remove gene regions with incongruent phylogenetic signals for several genes widely used in plant systematics. Here, I implement such a method that is available in a user friendly web interface.     

2-D graphical representation for characteristic sequences of DNA and its application

DNA sequencing has resulted in an abundance of data on DNA sequences for various species. Hence, the characterization and comparison of sequences become more important but still difficult tasks. In this paper, we first give a 2-D ladderlike graphical representation for the characteristic sequences of a DNA sequence, and then construct a 3-component vector, in which the normalized ALE-indices extracted from such three 2-D graphs via D/D matrices are individual components, to characterize the DNA sequence. The examination of similarities/dissimilarities among sequences of the beta-globin genes of different species illustrates the utility of the approach.     

A new method for DNA surface-property display and its application to E. coli promoter sequences

A personal computer program to visualize and compare the property of double-stranded DNA surface has been developed. Comparison of the surface property between Watson-Crick base-pairs in B-form DNA has elucidated that the base-pair replacement between a "degenerated base-pairs" conserves the pattern of potential hydrogen-bonding sites in both major and minor grooves. The idea of the "degenerated base-pairs" was applied for the problem of the base-sequence variation from the consensus sequence in the -35 region of E. coli promoter. The sequence variation is found to have tendency to occur among the degenerated base-pairs.     

DNA engineering and its application to nanotechnology.

The combination of branched DNA molecules and 'sticky' ends creates a powerful molecular assembly kit for structural DNA nanotechnology. Polyhedra, complex topological objects, a nanomechanical device and two-dimensional arrays with programmable surface features have already been produced in this way. Future applications range from macromolecular crystallography and new materials to molecular electronics and DNA-based computation.     

PNN-curve: a new 2D graphical representation of DNA sequences and its application

We introduce a novel 2D graphical representation of DNA sequences based on the pairs of the neighboring nucleotides (PNNs). Then we get the PNNs' distributions and obtain a y-M. The construction of the PNN-curve has some important advantages (1) It avoids loss of information and the PNN-curve standing for DNA sequences does not overlap or intersect with itself. (2) The novel 2D representation is more sensitive. The utility of this method can be illustrated by the examination of similarities/dissimilarities among the coding sequences of the first exon of beta-globin gene of eleven different species in Table 2.     

Application of a degenerate consensus sequence to quantify recognition sites by vertebrate DNA topoisomerase II

A consensus sequence has been derived for vertebrate topoisomerase II cleavage of DNA (Spitzner, J. R. and Muller, M. T. (1988) Nucleic Acid. Res. 16, 5533-5556). An independent sample of 65 topoisomerase II sites (obtained in the absence of topoisomerase II inhibitors) was analyzed and found to match the consensus sequence as well as enzyme sites determined in the presence of the anti-tumor drug 4'-(9-acridinyl-amino)-methanesulfon-m-anisidide (m-AMSA). As originally described, conventional application of the consensus sequence afforded accuracy in the prediction of the locations but not the frequencies of topoisomerase II cleavages. In the present report, we describe a new method which quantitatively discriminates sites from nonsites, called the 'matrix mean' method (the mean match of a site to the matrix of base proportions from the original consensus sequence derivation). Furthermore, we derived a second method, called the 'unique score' model, which predicts frequency of topoisomerase II activity at a cleavage site. In the unique score method both DNA strands of a site are examined to determine the total number of the consensus positions that match on at least one strand of a potential site. From the new data base of 65 topoisomerase II sites, cleavages were scored for relative cleavage strength. Linear regression analysis showed a significant (p less than 0.01) correlation between the unique score and cleavage strength. The study was extended to show that the unique score model accurately and quantitatively predicts topoisomerase II sites either in the absence or presence of m-AMSA using the same consensus sequence.     

A simple and efficient amplification method of DNA with unknown sequences and its application to microdissection/microcloning.

An alternative method for amplification of DNA with unknown sequences was developed. This involves the direct ligation of a primer oligodeoxyribonucleotide itself to restricted DNA fragments with unknown sequences to be amplified by PCR. The oligonucleotide need not be phosphorylated and need not be annealed with its complementary oligonucleotide in advance for ligation. The ligation reaction seems to be independent of the concentration of unknown DNA, proceeds in short time, and is efficient. The ligation efficiency was more than 30% at a low concentration, 10 fg/microliters, of DNA. This method was applied to a microdissection/microcloning of the short arm of human chromosome 2. Of 65 clones screened for the highly repetitive sequences with total human genomic DNA, eleven (17%) were positive. Their inserts ranged in size from 150 to 1,200 bp (average, 460 bp). In Southern blot analysis, thirty consecutive clones all detected signals common to both total human genomic DNA and mouse-human hybrid cell DNA containing only chromosome 2 of human origin. Among them, 24 (80%) were unique sequences, and 6 (20%) were multi-copy (or intermediate-repeat) sequences. Thus, this method is simple and efficient, and provides an alternative way to amplify unknown DNA.     

To clone or not to clone: method analysis for retrieving consensus sequences in ancient DNA samples

The challenges associated with the retrieval and authentication of ancient DNA (aDNA) evidence are principally due to post-mortem damage which makes ancient samples particularly prone to contamination from "modern" DNA sources. The necessity for authentication of results has led many aDNA researchers to adopt methods considered to be "gold standards" in the field, including cloning aDNA amplicons as opposed to directly sequencing them. However, no standardized protocol has emerged regarding the necessary number of clones to sequence, how a consensus sequence is most appropriately derived, or how results should be reported in the literature. In addition, there has been no systematic demonstration of the degree to which direct sequences are affected by damage or whether direct sequencing would provide disparate results from a consensus of clones.To address this issue, a comparative study was designed to examine both cloned and direct sequences amplified from ～3,500 year-old ancient northern fur seal DNA extracts. Majority rules and the Consensus Confidence Program were used to generate consensus sequences for each individual from the cloned sequences, which exhibited damage at 31 of 139 base pairs across all clones. In no instance did the consensus of clones differ from the direct sequence. This study demonstrates that, when appropriate, cloning need not be the default method, but instead, should be used as a measure of authentication on a case-by-case basis, especially when this practice adds time and cost to studies where it may be superfluous.     

Pro-Frame: similarity-based gene recognition in eukaryotic DNA sequences with errors

Performance of existing algorithms for similarity-based gene recognition in eukaryotes drops when the genomic DNA has been sequenced with errors. A modification of the spliced alignment algorithm allows for gene recognition in sequences with errors, in particular frameshifts. It tolerates up to 5% of sequencing errors without considerable drop of prediction reliability when a sufficiently close homologous protein is available (normalized evolutionary distance similarity score 50% or higher).