Single Tube, High Throughput Cloning of Inverted Repeat Constructs for Double-Stranded RNA Expression

BACKGROUND: RNA interference (RNAi) has emerged as a powerful tool for the targeted knockout of genes for functional genomics, system biology studies and drug discovery applications. To meet the requirements for high throughput screening in plants we have developed a new method for the rapid assembly of inverted repeat-containing constructs for the in vivo production of dsRNAs. METHODOLOGY/PRINCIPAL FINDINGS: The procedure that we describe is based on tagging the sense and antisense fragments with unique single-stranded (ss) tails which are then assembled in a single tube Ligase Independent Cloning (LIC) reaction. Since the assembly reaction is based on the annealing of unique complementary single stranded tails which can only assemble in one orientation, greater than ninety percent of the resultant clones contain the desired insert. CONCLUSION/SIGNIFICANCE: Our single-tube reaction provides a highly efficient method for the assembly of inverted repeat constructs for gene suppression applications. The single tube assembly is directional, highly efficient and readily adapted for high throughput applications

Annealing as visualized by agarose gel electrophoresis.

<p>Gel image showing the annealing of three different Gl1 RNAi constructs The sense, antisense and annealed products are labeled S, AS and Annealed respectively The sense product is larger than the antisense product since it is tethered to the 150 bp universal loop The sizes of the relevant size markers are indicated.</p

Rice (Oryza sativa) centromeric regions consist of complex DNA

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence in situ hybridization. Six of the elements are middle repetitive, and their copy numbers range from ≈50 to ≈300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented ≈6,000 times in the rice genome and is detected only in Oryza species. Fiber-fluorescence in situ hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and Arabidopsis thaliana chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences

LIC cloning of inverted repeats into three different vectors.

<p>Successful cloning is based on the availability of the desired target sequences, so failure to amplify the target sequence is a PCR failure and is not considered a cloning failure. The cloning success rate refers to the number of the desired LIC constructs attained following one round of cloning; this includes incorporation of the uracil containing primers, addition of the loop, and generation of the ss tails and annealing to the vector in the desired orientation.</p

Strategy for LIC-based assembly of palindromic RNAi constructs.

<p>Four PCR primers are synthesized containing the s5′ and a3′ tags tethered to the forward primer, and the a5′ and s3′ tags tethered to the reverse primer For each target two amplifications are carried out using the a5′/a3′ primer pair and the s5′/s3′ pair to generate the antisense and sense products respectively In the second round of PCR an aliquot of each reaction is amplified using the cognate dU containing primers During the second round of PCR a universal loop is added to the sense product by SOE-PCR The loop is prepared by PCR amplification and a stock is stored at −20°C For the LIC based assembly 5 µl aliquots are mixed with 10 ng of vector, treated with UDG, heated to 65°C and allowed to anneal.</p

Universal loop facilitates DNA sequence confirmation of the insert.

<p>DNA sequence of the universal loop is shown in panel A Panel B depicts the restriction sites used to linearize the construct for sequence confirmation. The relative position of the sequencing primers is depicted and their sequences are provided.</p

Primers for LIC-based assembly.

<p>PCR primers used for the assembly of palindromic RNAi constructs The first round PCR sequences correspond to the 5′ tags with are tethered to the gene specific primer sequences The second round PCR primers contain dU residues used to generate ss tails The vector overhang depicts the single stranded regions of the vector which are generated following HpaI and NbBbvCI digestion The left and right ss vector overhangs are complementary to the 3′ tails generated by UDG cleavage of dUa5′ and dUs3′ of their respective PCR products.</p

A conserved repetitive DNA element located in the centromeres of cereal chromosomes

Repetitive DNA sequences have been demonstrated to play an important role for centromere function of eukaryotic chromosomes, including those from fission yeast, Drosophila melanogaster, and humans. Here we report on the isolation of a repetitive DNA element located in the centromeric regions of cereal chromosomes. A 745-bp repetitive DNA clone, pSau3A9, was isolated from sorghum (Sorghum bicolor). This DNA element is located in the centromeric regions of all sorghum chromosomes, as demonstrated by fluorescence in situ hybridization. Repetitive DNA sequences homologous to pSau3A9 also are present in the centromeric regions of chromosomes from other cereal species, including rice, maize, wheat, barley, rye, and oats. Probe pSau3A9 also hybridized to the centromeric region of B chromosomes from rye and maize. The repetitive nature and its conservation in distantly related plant species indicate that the pSau3A9 family may be associated with centromere function of cereal chromosomes. The absence of DNA sequences homologous to pSau3A9 in dicot species suggests a faster divergence of centromere-related sequences compared with the telomere-related sequences in plants

Functional rice centromeres are marked by a satellite repeat and a centromere-specific retrotransposon

The centromere of eukaryotic chromosomes is essential for the faithful segregation and inheritance of genetic information. In the majority of eukaryotic species, centromeres are associated with highly repetitive DNA, and as a consequence, the boundary for a functional centromere is difficult to define. In this study, we demonstrate that the centers of rice centromeres are occupied by a 155-bp satellite repeat, CentO, and a centromere-specific retrotransposon, CRR. The CentO satellite is located within the chromosomal regions to which the spindle fibers attach. CentO is quantitatively variable among the 12 rice centromeres, ranging from 65 kb to 2 Mb, and is interrupted irregularly by CRR elements. The break points of 14 rice centromere misdivision events were mapped to the middle of the CentO arrays, suggesting that the CentO satellite is located within the functional domain of rice centromeres. Our results demonstrate that the CentO satellite may be a key DNA element for rice centromere function