Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.     

A consensus sequence for binding of SmcR, a Vibrio vulnificus LuxR homologue, and genome-wide identification of the SmcR regulon

Quorum sensing has been implicated as an important global regulatory system controlling the expression of numerous virulence factors in bacterial pathogens. In the present study, DNA targets of SmcR, a Vibrio vulnificus LuxR homologue, were selected from a random pool of DNA fragments by using a cycle selection procedure consisting of in vitro DNA-SmcR interaction, purification of SmcR-DNA complexes, and PCR amplification of SmcR-bound DNA. The amplified DNA fragments were cloned and analyzed separately by electrophoretic mobility shift assay to verify the specific binding of SmcR to the DNA. The DNA sequences bound by SmcR were determined by DNase I footprinting, and alignment of the resulting 29 sequences revealed a 22-bp consensus SmcR-binding sequence, 5'-TTATTGATWWRWTWNTNAATAA-3' (where W represents A or T, R is G or A, and N is any nucleotide), with an 8-bp (TTATTGAT) inverted repeat. The consensus sequence revealed greater efficiency for the binding of SmcR than the SmcR-binding sequence previously identified within P(vvpE). Mutational analysis demonstrated that the 9th and 10th bases from the center are the most essential for SmcR binding. A genome-wide search using the consensus sequence predicted that at least 121 genes are under the control of SmcR, and 10 of these newly identified SmcR regulon members were verified as being regulated by SmcR in V. vulnificus as well as in vitro. The consensus sequence and newly identified genes should be of use for elucidating the regulatory mechanism of SmcR and provide further insight into the role of the quorum sensing in V. vulnificus pathogenesis.     

Non-SELEX: selection of aptamers without intermediate amplification of candidate oligonucleotides

Aptamers are typically selected from libraries of random DNA (or RNA) sequences through systematic evolution of ligands by exponential enrichment (SELEX), which involves several rounds of alternating steps of partitioning of candidate oligonucleotides and their PCR amplification. Here we describe a protocol for non-SELEX selection of aptamers--a process that involves repetitive steps of partitioning with no amplification between them. Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), which is a highly efficient affinity method, is used for partitioning. NECEEM also facilitates monitoring of bulk affinity of enriched libraries at every step of partitioning and screening of individual clones for their affinity to the target. NECEEM allows all clones to be screened prior to sequencing, so that only clones with suitable binding parameters are sequenced. The entire protocol can be completed in 1 wk, whereas conventional SELEX protocols take several weeks even in a specialized industrial facility.     

A method to select chemically modified aptamers directly

In vitro selection is a strategy to identify high-affinity ligands of a predetermined target among a large pool of randomized oligonucleotides. Most in vitro selections are performed with unmodified RNA or DNA sequences, leading to ligands of high affinity and specificity (aptamers) but of very short lifetime in the ex vivo and in vivo context. Only a very limited number of modified triphosphate nucleotides conferring nuclease resistance to the oligomer can be incorporated by polymerases. This encourages the development of alternative methods for the identification of nuclease-resistant aptamers. In this paper, we describe such a method. After selection of 2'O-methyl oligonucleotides against the TAR RNA structure of HIV-1, the complementary DNA sequences are fished out of a pool of randomized oligodeoxynucleotides by Watson-Crick hybridization. The DNA-fished sequences are amplified by PCR as double and single strands, the latter being used to fish back the chemically modified candidates from the initial library. This procedure allows an indirect amplification of the selected candidates. This enriched pool of modified sequences is then used for the next selection round against the target.     

Selection of deletion mutants by polymerase chain reaction

Polymerase chain reaction (PCR) based DNA amplification has replaced many time-consuming protocols in molecular biology. Here we describe a simple strategy to quickly select deletion mutants based on PCR methodology which then can be confirmed by nucleotide sequencing. A forward PCR primer is designed in such a way to recognize only the wild type sequences in the amplification reaction and thus a negative selection identifies the deletion in the samples.     

Emulsion PCR: a high efficient way of PCR amplification of random DNA libraries in aptamer selection

Aptamers are short RNA or DNA oligonucleotides which can bind with different targets. Typically, they are selected from a large number of random DNA sequence libraries. The main strategy to obtain aptamers is systematic evolution of ligands by exponential enrichment (SELEX). Low efficiency is one of the limitations for conventional PCR amplification of random DNA sequence library in aptamer selection because of relative low products and high by-products formation efficiency. Here, we developed emulsion PCR for aptamer selection. With this method, the by-products formation decreased tremendously to an undetectable level, while the products formation increased significantly. Our results indicated that by-products in conventional PCR amplification were from primer-product and product-product hybridization. In emulsion PCR, we can completely avoid the product-product hybridization and avoid the most of primer-product hybridization if the conditions were optimized. In addition, it also showed that the molecule ratio of template to compartment was crucial to by-product formation efficiency in emulsion PCR amplification. Furthermore, the concentration of the Taq DNA polymerase in the emulsion PCR mixture had a significant impact on product formation efficiency. So, the results of our study indicated that emulsion PCR could improve the efficiency of SELEX.     

Target Detection Assay (TDA): a versatile procedure to determine DNA binding sites as demonstrated on SP1 protein.

We developed a rapid method designated Target Detection Assay (TDA) to determine DNA binding sites for putative DNA binding proteins. A purified, functionally active DNA binding protein and a pool of random double-stranded oligonucleotides harbouring PCR primer sites at each end are included the TDA cycle which consists of four separate steps: a DNA protein incubation step, a protein DNA complex separation step, a DNA elution step and a polymerase chain reaction (PCR) DNA amplification step. The stringency of selection can be increased in consecutive TDA cycles. Since tiny amounts of retained DNA can be rescued by PCR, buffer systems, salt concentrations and competitor DNA contents can be varied in order to determine high affinity binding sites for the protein of choice. To test the efficiency of the TDA procedure potential DNA binding sites were selected by the DNA binding protein SP1 from a pool of oligonucleotides with random nucleotides at 12 positions. Target sites selected by recombinant SP1 closely matched the SP1 consensus site. If DNA recognition sites have to be determined for known, mutated or putative DNA binding proteins, the Target Detection Assay (TDA) is a versatile and rapid technique for consideration.     

A simulation of subtractive hybridization.

Various strategies employed in genomic DNA cloning by subtractive hybridization have been examined by computer simulations, with the comparison between the predictions and the published results. The result shows that the efficiency of target sequence enrichment and the sensitivity to experimental conditions depend strongly on the enrichment strategy employed. The strategy selecting only tester/tester after hybridization can be very efficient to enrich targets. For successful target enrichment, however, the strategy requires a highly efficient subtraction method and proper hybridization conditions. The strategy also requires that the selected DNA be amplified by polymerase chain reaction (PCR) after each or each alternate subtraction. By contrast, the strategy selecting tester/tester plus single-stranded tester is less sensitive to various experimental factors, compared with the strategy selecting only tester/tester. However, it is not as efficient. With this strategy, the tester DNA selected may or may not be amplified by PCR before the next round. In the case of the strategy selecting single-stranded tester, the target DNA can be successfully enriched only when the selected DNA is directly used without PCR amplification in the next round. The strong features of existing methods can be combined to develop a protocol that is more efficient and more reliable.     

A role in DNA binding for the linker sequences of the first three zinc fingers of TFIIIA.

Zinc fingers of the TFIIIA type are connected by short linker sequences between the structural units. Structural investigations by 2D NMR in solution and by X-ray crystallographic analyses of complexes with DNA point to a passive role for the linkers. We have therefore investigated the influence of the linker sequence on DNA binding using as a model the first three fingers of the protein TFIIIA. Insertion of certain heterologous linkers abolishes binding, and replacement of individual amino acids can reduce binding by factors of up to twenty-four.     

Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR

Locked nucleic acid (LNA) is a modified DNA with increased binding affinityfor complementary DNA sequences. Our strategy was to use this property of LNA to inhibit undesired PCR amplification (e.g.,from contaminating genomic DNA) in a cDNA-based assay. By placing a short complementary LNA sequence in intronic DNA, the aim was to inhibit the amplification of genomic DNA without affecting the amplification of reverse-transcribed spliced mRNA. LNA was designed to bind within intron 5 in the x-box binding protein 1 (XBP1) gene. An irrelevant LNA oligonucleotide served as a negative control. In both PCR and real-time PCR, the addition of LNA showed blocking of the amplification of genomic XBP1 but not cDNA XBP1. To test the effect of melting temperature (Tm) on the LNA, we investigated the number of LNA nucleotides that could be replaced with DNA nucleotides and still retain the blocking activity. More than three DNA nucleotides reduced the LNA inhibition ability. The sequence specificity of the LNA was tested by investigating the number of LNA nucleotide mismatches permitted. The introduction of one mismatch maintained the inhibition of genomic amplification whereas two mismatches reduced the amplification. Our results show that LNA may be used to enhance the specificity of PCR by eliminating unwanted PCR products.     

Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder.

The tripeptide 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder (MGB) is conjugated to the 5'-end of short oligodeoxynucleotides (ODNs), the conjugates form unusually stable hybrids with complementary DNA in which the tethered CDPI3group resides in the minor groove. We show that these conjugates can be used as PCR primers. Due to their unusually high binding affinity, conjugates as short as 8-10mers can be used to amplify DNA with good specificity and efficiency. The reduced length primers described here might be appropriate for the PCR amplification of viral sequences which possess a high degree of variability (e.g., HPV, HIV) or for recent techniques such as gene hunting and differential display which amplify multiple sequences using short primer pairs.     

Rapid isolation of flanking genomic DNA using biotin-RAGE, a variation of single-sided polymerase chain reaction.

A method is described for quickly and reproducibly isolating genomic DNA contiguous with known DNA sequence by means of the polymerase chain reaction (PCR). Flanking genomic DNA is isolated using a biotinylated sequence-specific primer in combination with a generic hybrid primer that binds to a deoxyoligonucleotide sequence artificially added to the ends of the genomic DNA. Amplified sequences that include the biotinylated primer are purified from nonbiotinylated amplification products by binding to a solid-phase streptavidin matrix. The biotinylated amplification product(s) are subjected to a further round of amplification, after which they can be subcloned and analyzed. This technique was applied to the isolation of three intron-exon junctions. Verification of the identify of these junction sequences was accomplished by designing primers based on the intron sequences isolated by Biotin-RAGE, amplifying across the exon using these intron primers, and sequencing the PCR-generated product.     

cDNA selection with YACs

Identification of expressed sequence tags (ESTs) in large genomic segments is an important step in positional cloning and genomic mapping studies. A simple and efficient polymerase chain reaction (PCR)-based approach is described here to identify coding sequences in large genomic fragments of DNA cloned in vectors such as yeast artificial chromosome (YAC) vectors. The method is based on blocking of sequences such as repetitive and GC rich sequences in the genomic DNA immobilized on nylon paper discs prior to hybridization of the discs to cDNA library, and recovery of the selected cDNAs by the PCR. Single or multiple cDNA libraries can be used in the selection procedure. The procedure has been used successfully also with total yeast DNA containing a YAC.     

A strategy to study gene polymorphism by direct sequence analysis of cosmid clones and amplified genomic DNA

A two-step strategy is described here to rapidly analyze gene-sequence variation or polymorphism. First, DNA sequences flanking the coding region of the gene to be analyzed are determined directly from a cosmid clone, including the gene, using the modified T7 DNA polymerase and sequencing primers based on the cDNA sequence of the gene. Second, the identified gene-flanking sequences are used to design amplification primers for the polymerase chain reaction (PCR) to permit amplification of DNA segments of up to 1 kilobase in genomic DNA from multiple individuals. These amplified DNA segments are directly sequenced using the thermostable Taq DNA polymerase.     

One multiplex control for 29 cystic fibrosis mutations

A simple approach is described to synthesize and clone an inexhaustible supply of any homozygous and/or heterozygous controls diluted with yeast genomic DNA to mimic human genome equivalents for use throughout the entire multiplex mutation assay. As a proof of principle, the 25 cystic fibrosis mutation panel selected by the American College of Medical Genetics and four additional mutant sequences were prepared as a single control mixture. The 29 CFTR mutations were incorporated into 17 gene fragments by PCR amplification of targeted sequences using mutagenic primers on normal human genomic DNA template. Flanking primers selected to bind beyond all published PCR primer sites amplified controls for most assay platforms. The 17 synthesized 433-933-bp CFTR fragments each with one to four homozygous mutant sequences were cloned into nine plasmid vectors at the multiple cloning site and bidirectionally sequenced. Miniplasmid preps from these nine clones were mixed and diluted with genomic yeast DNA to mimic the final nucleotide molar ratio of two CFTR genes in 6 x 10(9) bp total human genomic DNA. This mixture was added to control PCR reactions prior to amplification as the only positive control sample. In this fashion >200 multiplex clinical PCR analyses of >4,000 clinical patient samples have been controlled simultaneously for PCR amplification and substrate specificity for 29 tested mutations without cross contamination. This clinically validated multiplex cystic fibrosis control can be modified readily for different test formats and provides a robust means to control for all mutations instead of rotating human genomic controls each with a fraction of the mutations. This approach allows scores of additional mutation controls from any gene loci to be added to the same mixture annually.     

A simple and efficient method to determine the terminal sequences of restriction fragments containing known sequences.

We present an improvement of the inverse PCR method for the determination of end sequences of restriction fragments containing unknown DNA sequences flanked by known segments. In this approach, a short "bridge" DNA is inserted during the self-ligation step of the inverse PCR technique. This bridge DNA acts as primer annealing sites for amplification and subsequent direct sequencing. Successive PCR amplifications enable selective amplification of the unknown sequences from a complex mixture. Unlike previously described methods, our method does not require special materials, such as synthetic adapters or biotinylated primers that must be prepared each time to adapt the target. Furthermore, no complex steps such as dephosphorylation or purification are needed. Our method can save time and reduce the cost of cloning unknown sequences; it is ideal for routine, rapid gene walking. We applied this method to a GC-rich bacterial genome and succeeded in determining the end sequences of a 4.5-kb fragment.     

Minimal primer and primer-free SELEX protocols for selection of aptamers from random DNA libraries

Standard systematic evolution of ligands by exponential enrichment (SELEX) protocols require libraries that contain two primers, one on each side of a central random domain, which allow amplification of target-bound sequences via PCR or RT-PCR. However, these primer sequences cause nonspecific binding by their nature (generally adding about 20 nt on each end of the random sequence of about 30-40 nt), and can result in large numbers of false-positive binding sequences and/or interfere with good binding random sequences. Here, we have developed two DNA-based methods that reduce and/or eliminate the primer sequences from the target-binding step, thus reducing or eliminating the interference caused by the primer sequences. In these methods, the starting selection libraries contain a central random sequence that is: (i) flanked by only 2 nt on each side (minimal primer); or (ii) flanked only by either a 2- or 0-nt overhand on the 3' end (primer-free). These methods allow primer regeneration and re-elimination after and before selection, are fast and simple, and don't require any chemical modifications for selection in a variety of conditions. Further, the selection rounds are performed with DNA oligomers, which are generally employed as end product aptamers.