Systematic comparison of gene expression through analysis of cDNA fragments within or near to the protein-coding region.

Life is controlled by the timely and ordered expression of genes. Identification of important genes involved in specific physiological and pathological conditions requires efficient methods to analyse differential gene expression. We describe a novel strategy, namely complete comparison of gene expression (CCGE), for a systematic assessment of differentially expressed genes. Using the CCGE method, double-stranded cDNA is digested with two restriction enzymes that cut with different frequencies, the representative cDNA fragments are generated within or near to the protein-coding region. After being flanked by two different types of adapters, and amplified by a nested suppression PCR, the selected cDNA fragments, representing entire cDNA population, can be divided into 256 subsets; amplified and compared in a systematic manner.     

Molecular indexing of human genomic DNA

Molecular indexing sorts DNA fragments into subsets for inter-sample comparisons. Type IIS or interrupted palindrome restriction endonucleases, which result in single-stranded ends not including the original recognition sequence of the enzyme, are used to produce the fragments. The ends can then be any sequence but will always be specific for a given fragment. Fragments with particular ends are selected by ligation to a corresponding indexing adapter. We describe iterative indexing, a new process that after an initial round of indexing uses a Type IIS restriction endonuclease to expose additional sequence for further indexing. New plasmids, pINDnn, were produced for novel use as indexing adapters. Together, the plasmids index all 16 possible dinucleotides. Their large size can be increased by dimerisation in vitro and allows the isolation of indexed material by size separation. Fragments produced from human genomic DNA by Type II restriction endonucleases were sorted using six bases in total to a possible enrichment of 1920-fold. By comparison with the public human sequence databases, fidelity of indexing was shown to be high and was tolerant of repetitive sequences. Genome-wide comparisons on a candidate or non-candidate basis are made possible by this approach.     

Subtractive differential display: a modified differential display technique for isolating differentially expressed genes

Differential display (DD) is a novel PCR-based technique, very commonly used to study differentially expressed genes at the mRNA level. In this paper we report a modified version of this technique that we have used to study the differences between the mRNA population from brain tissue of adult and old rats. We have modified the technique to enhance reproducibility and reduce false positives and redundancy. It is fast and does not require any expensive or uncommon reagent. We choose to call it as subtractive differential display as it is a differential display performed over subtracted mRNA population. We have used this protocol successfully to clone a number of age-related differentially expressed sequences from rat brain that need to be sequenced to establish the gene identity.     

Cloning of region-specific genetic markers of planaria using a new method--ordered differential display

A new method for finding differentially expressed genes, termed ordered differential display of mRNAs (ODD), was used in the search for region-specific molecular markers of freshwater planarian Dugesia tigrina. In this method, the effect of selective suppression of a polymerase chain reaction (PCR) is used for the differential amplification of a pool of 3'-terminal cDNA fragments generated by digestion of cDNAs with a restriction endonuclease. In the resulting amplified cDNAs, every mRNA is represented by a cDNA fragment whose length is determined by the position of the restriction site nearest to the 3'-terminus. Subsequent PCR with primers 3'-extended by two random nucleotides allowed the amplification of 1/192 part of all cDNA molecules present in the sample. The comparison of the generated pools of cDNA molecules separated by PAGE leads to the identification of differentially expressed sequences. The systematic study of the total mRNA pool is achieved by the successive use of all possible combinations of extended primers. Some sequences preferentially expressed along the anterior-posterior axis of planarian were identified using ODD.     

Identification of differentially expressed genes by restriction endonuclease-based gene expression fingerprinting.

A novel method for identification of differentially expressed genes has been developed. It is based on the consecutive restriction digestions of 3' terminal cDNA fragments to produce a fingerprint of gene expression. cDNA molecules are synthesized using a biotinylated oligo(dT) primer, digested with a frequently cutting restriction endonuclease and the 3'-terminal restriction fragments are isolated using streptavidin microbeads. After amplification by PCR, cDNA fragments are immobilized again on streptavidin beads, radiolabeled and treated sequentially with a set of restriction endonucleases. The products of individual enzymatic reactions from two or more different RNA populations are resolved by polyacrylamide gel electrophoresis and compared to reveal differentially expressed genes. This strategy enabled us to identify and clone the fragments of five genes expressed differentially in murine thymus and spleen. One of the genes was found to encode terminal deoxynucleotidyl transferase; others are apparently previously unknown genes.     

Rapid selection of differentially expressed genes in TNF[alpha]-activated endothelial cells

BACKGROUND: RNA differential display (DD) RT-PCR is a useful method to identify and clone differentially expressed genes. However, the rate of false positives and redundancy associated with this PCR-based method as well as laborious downstream screening steps constitute major limitations.Here we present DD RT-PCR and reverse northern (RN) protocols allowing rapid and acurate identification of genes upregulated in porcine endothelial cells (EC) in response to TNFalpha. MATERIALS AND METHODS: The housekeeping gene beta-actin was used to investigate mispriming and to set up optimal conditions for DD-RT-PCR and RN. In this study DD was performed to compare resting and TNFalpha-activated ECs. Selection of DD-fragments was performed following 30-cycles of PCR using serial dilutions of template cDNA and regulation of 6 out of 17 candidates genes were first confirmed by semi-quantitative RN. RESULTS: Using this protocol, 5 out of 6 DD-fragments were further confirmed to be upregulated by Northern blot, and 3 novel porcine cDNAs were cloned including the pro-apoptotic member of the Bcl-2 family, Noxa. CONCLUSION: In this study we demonstrate that the combination of DD-RT-PCR and RN, which efficiently reduces the number of false positive candidates derived from mispriming at the screening step, allows a rapid identification of differentially expressed genes.     

A modified cDNA subtraction to identify differentially expressed genes from plants with universal application to other eukaryotes

We have designed a simple and efficient polymerase chain reaction (PCR)-based cDNA subtraction protocol for high-throughput cloning of differentially expressed genes from plants that can be applied to any experimental system and as an alternative to DNA chip technology. Sequence-independent PCR-amplifiable first-strand cDNA population was synthesized by priming oligo-dT primer with a defined 5' heel sequence and ligating another specified single-stranded oligonucleotide primer on the 3' ends of first-strand cDNAs by T4 RNA ligase. A biotin label was introduced into the sense strands of cDNA that must be subtracted by using 5' biotinylated forward primer during PCR amplification to immobilize the sense strand onto the streptavidin-linked paramagnetic beads. The unamplified first strand (antisense) of the interrogating cDNA population was hybridized with a large excess of amplified sense strands of control cDNA. We used magnetic bead technology for the efficient removal of common cDNA population after hybridization to reduce the complexity of the cDNA prior to PCR amplification for the enrichment and sequence abundance normalization of differentially expressed genes. Construction of a subtracted and normalized cDNA library efficiently eliminates common abundant cDNA messages and also increases the probability of identifying clones differentially expressed in low-abundance cDNA messages. We used this method to successfully isolate differentially expressed genes from Pennisetum seedlings in response to salinity stress. Sequence analysis of the selected clones showed homologies to genes that were reported previously and shown to be involved in plant stress adaptation.     

Isolation of developmentally regulated genes by differential display screening of cDNA libraries.

mRNA differential display RT-PCR has been extensively used for the isolation of genes differentially expressed between RNA populations. We have assessed its utility for the identification of developmentally regulated genes in plasmid cDNA libraries derived from individual tissues dissected from early mouse embryos. Using plasmid Southern blot hybridisation as a secondary screen, we are able to identify such genes and show by whole-mount in situ hybridisation that their expression pattern is that expected from the differential display profile.     

Creation of a type IIS restriction endonuclease with a long recognition sequence

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.     

A simple, effective method for the construction of subtracted cDNA libraries

A simple method is described for the construction of subtracted cDNA libraries. The technique was used to create a human pancreatic tumor cDNA library that was screened using either hybridization with cDNA probes or antibodies. cDNA from a well-differentiated tumor cell line (CD-11) was subtracted against RNA from an undifferentiated tumor cell line (Panc-1). The subtracted cDNA was purified from RNA-cDNA hybrids by oligo-dA cellulose affinity chromatography. Single-stranded subtracted cDNA was used as a template for random primed second-strand synthesis using the Klenow's fragment of DNA polymerase. After ligation with Eco R1 adapters, cDNA was inserted into lambda gt11. A library of 140,000 primary pfu was obtained that contained 92% recombinants. A small portion of this library (40,000 pfu) was subjected to probe screening with a mucin cDNA probe known to be differentially expressed by CD-11 cells. The ratio of mucin cDNA clones to actin cDNA clones was increased by greater than 300-fold in the subtracted cDNA library compared to a standard cDNA library from the same cell line. The absolute number of mucin cDNA clones per 40,000 pfu was also increased 32-fold in the subtracted library. Pancreatic tumor mucin cDNAs were also identified in the subtracted library by antibody screening. The subtraction procedure yielded a 50-fold enrichment in differentially expressed cDNA detected by antibodies, compared to a nonsubtracted library from the same cell line.     

A statistical model providing comprehensive predictions for the mRNA differential display

MOTIVATION: Differential display (DD) or arbitrarily primed fingerprinting serves to identify differentially expressed genes, but these techniques cannot determine how many of the theoretically available genes have been uncovered. Previous mathematical models are unsatisfying as they are not suitable to analyze experimental data. RESULTS: In the present study, we provide a statistical model based on the redundancy of cDNA fragments amplified during DD experiments. This model is applicable to any DD and predicts (1) the total number of genes expressed in a sample cell type or tissue, (2) the number of differentially expressed genes, (3) the coverage obtained with any given number of primer combinations. In a DD experiment comparing two developmental stages of the post natal rat inner ear, we estimated the total number of differentially expressed genes accessible by DD to be 445, and the number of primer combinations required to uncover 90% of these to be 127. AVAILABILITY: The algorithms were implemented in Matlab (The Mathworks, Inc., Natick, MA) environment and are available at www.physiologie.uni-freiburg.de/download.html CONTACT: ellen.reisinger@physiologie.uni-freiburg.de.     

Use of primer-restriction-end adapters in a novel cDNA cloning strategy

We introduce a class of synthetic oligonucleotides, referred to as primer-restriction-end (PRE) adapters, which are bifunctional, one end serving as a primer for a polymerase reaction, while the other end can be ligated to restriction endonuclease digested DNA. Use of such adapters forms the basis of a new method for inserting single-stranded cDNA into cloning vectors, which involves very few separate biochemical modifications of the cDNA and so is appropriate when extensive fractionation of cDNA is desired prior to cloning. This novel methodology is highly efficient in producing full-length cDNA cloned in a predictable orientation within vectors, as we demonstrate by constructing and analysing clones of immunoglobulin lambda light chain cDNA in Escherichia coli.