This protocol describes collection of massive data from protein-DNA binding microarray for primase, an enzyme that transiently binds to specific DNA sequences and in turn catalyzes formation of RNA primers. This video will cover how microarray technology can help redefining primase sequence recognition sites and how a high throughput approach can compliment classic biochemistry. This technique combines two approaches:primase DNA binding microarray and primase activity assay.
The microarray provides massive data of primase binding to DNA sequences, where as the biochemical assay provides information on RNA primer formation by DNA primase. The tested enzymatic activity depends on the insight from the microarray experiment and its lower throughput in its nature. The link between the efficiency of binding, DNA sequence selection and the activity of the enzyme is probed.
Identification of sequence determinance using microarray, allows us to draw accurate conclusions based on massive data of the microarray. This approach has been used so far to describe DNA binding sequences of transcription factors. Transcription factors are static proteins that bind tightly to DNA.
Demonstrating the procedure will be Stefan Ilic. To begin this procedure, pre wet the microarray slide by placing it in a coplin jar containing 0.01%Triton-X 100. And rotating on a lab rotator, add 125 rpm for five minutes.
Pipette the blocking solution into a plastic box. Then remove the microarray slide from the coplin jar. Use a fine wipe to drive the non-DNA side and edges of the slide.
Slowly place the microarray into the plastic box. Incubate at room temperature for one hour with slow shaking. After this, wash the slide once with 0.01%Tween-20 in PBS.
On a lab rotator at 125 rpm for five minutes. Remove the Tween-20 and rinse the slide once with 0.01%Triton-X 100 in PBS on a lab rotator at 125 rpm for two minutes. Then quickly transfer the slide to a coplin jar containing PBS.
First, assemble the PMB chamber as outlined in the text protocol. Place the slide into the designated space. Use tweezers to push it down and left.
Place the silicon gasket on top, making sure that it is well aligned with the lower part of the PBM chamber. Then close the chamber and tighten the screws diagonally. Pipette the protein binding mixture into each well of the gasket.
Then, incubate at room temperature for 30 minutes. To begin, pipette 0.05%Tween-20 in PBS into the PBM chamber to briefly wash the slide. Using a vacuum aspirator, remove the solution from the wells of the chamber being careful to not touch the DNA spots.
Repeat this, briefly wash the slide with PBS. And use the vacuum to remove the solution from the wells of the chamber while being careful to not touch the DNA spots. Next, add Alexa 488 conjugated anti-His Antibody to each well of the PBM chamber.
Incubate in the dark at room temperature for 30 minutes. After this, briefly wash the slide inside the PBM chamber by adding a few drops of 0.05%Tween-20 in PBS into each well. And use the vacuum aspirator to remove the solution from the wells of the chamber, being careful to not touch the DNA spots.
Disassemble the PBM chamber and remove the slide. Rinse the slides twice in 0.05%Tween-20 in PBS, with each rinse lasting three minutes. Then, rinse the slides two times in PBS, with each rinse lasting three minutes each.
And one time in double distilled water for three minutes. Dry the slides with compressed air. And store them in a dark slide box until ready to scan.
When ready, use a microarray scanner to scan the chip with a excitation of 495 nanometers and a emission of 19 nanometers. And collect the median fluorescence intensity. In this study pi throughput primase profiling is used to map the primase binding sites, including those that are difficult if not impossible to observe using classical tools.
Importantly, pi throughput primase profiling enables the revisiting of the traditional understanding of primase binding sites. Especially pi throughput primase profiling reveals binding specificities in addition to known 5'GTC3'recognition sequences, which leads to changes in functional activities of T7 DNA primase. Namely, two groups of sequences are identified.
Strong binding DNA sequences that contained TG in the flanks and weak binding DNA sequences that contained AG in the flanks. No primase binding to DNA templates that were missing 5'GTC3'within their sequences was detected. The primase DNA recognition sites that contains specific features such as TG rich flanks, increase primase DNA binding up to 10 fold.
Surprisingly, they also increase the length of newly formed RNA. Importantly, high throughput primase profiling allows us to observe and quantify the variability in primer length in relation to the sequence of the DNA template. When performing this procedure the washing steps should be subtle.
And buffers should contain components that logged the primase on the DNA. Also a threshold of futile binding events is determined biochemically. Therefore analysis of the microarray is insufficient.
Methods such as a surface plasma resonance and gel shift assays can determine the binding affinity of primase to select the DNA sequences. My lab implements machine learning prediction models to detect primase sequence determinants that yield productive primers that can be extended by DNA polymerase. In the era of big data, an implementation of statistical methods to analyze big data, the technique will definitely help to better characterize specific DNA sequences and link the sequence recognition to the activity of primase.
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