This method can detect the activity of retrotranspositions called LINE-1s. These mobile genetic elements cause de novo insertions in cancer genomes and thereby contribute to genomic instability. The advantage of this PCR-based technique is that it is simple and cost-effective compared to high-throughput sequencing approaches.
We demonstrate here its utility in detecting de novo insertions that originate from a LINE-1 inside the TTC28 gene on human chromosome 22. LINE-1 activity can serve as biomarker for malignancy or pre-malignancy especially in epithelial tumor types, like colon cancer or high-grade serous ovarian cancer. Besides LINE-1 insertions, this method can detect other genomic aberrations, such as DNA rearrangements that always need rearrangement for a reason.
In order to select a suitable restriction enzyme, and to design inverse primers, First, download the TTC28 LINE-1 sequence in FASTA format from a LINE-1 database, such as L1Base. The L1Base ID for TTC28 Line-1 is 135. Include five kilobases sequence, flanking both five prime and three prime, end of the Line-1 sequence.
Export the sequence to a word processor. Here the Line-1 flanking sequence is annotated in brown font and Line-1 sequences in gray font. To determine the unique tag into one kilobase sequence downstream of TTC28 Line-1's three prime and into one of the polyadenylation signal prediction tools, such as Polyadq or Dragon PolyA Spotter.
Annotate all polyadenylation signal in this one kilobase window. The sequence between TTC28 LINE-1's own weak polyadenylation signal highlighted here in pink and the strongest polyadenylation signal downstream is the unique tag of this LINE-1 highlighted here in yellow. To design inverse PCR primers enter the unique tag sequence into a web-based primer designing tool, like NCBI's Primer-BLAST that generates specific PCR primers, keep the product length parameter to minimum to show primer pairs are close to one another.
Select appropriate genome database. As the output of Primer Blast generates conventional PCR primers facing each other, use the reverse complement function for the primer pair to perform inverse PCR. Inter-design primers, corresponding to east polyadenylation signal whenever possible.
We have deigned three primer pairs, highlighted in teal and green, corresponding to three strong polyadenylation signal in the unique tag of TTC28 LINE-1. Next, to select a suitable restriction enzyme, digest the LINE-1 sequence, along with its five kilobase upstream and downstream flanks in silica using web-based tools, such as RestrictionMapper. This will give a comprehensive list of restriction enzymes that digests this reason, generating different restriction fragments.
Select restriction enzymes that make a five prime cut upstream of LINE-1's five prime end or towards the five prime end of the LINE-1 if it is within the LINE-1 itself. And a three prime card downstream up from the unique tag. The native restriction fragment with LINE-1 sequence and its unique tag made by selective restriction enzymes, should not be longer than 12 kilobases, as it might not be amplified by PCR efficiently.
Note that the selective restriction enzymes would be insensitive to DNA methylation, should be heat-inactivatable, and should generate sticky ends that are complementary to each other. In order to demonstrate long distance inverse PCR, we will use SacI restriction enzyme that cuts DNA at DAZCTC sites highlighted here in light green. SacI fulfills all the criteria at the TTC28 LINE-1 locus and generates a native restriction fragment of 5, 700 base pairs.
Extract genomic DNA from samples using commercially available DNA extraction kit that is able to extract good quality, high molecular DNA necessary for long distance inverse PCR. Here we have extracted genomic DNA from MCF7 breast cancer cell line and normal human blood using manufacture's instruction. Measure the DNA concentration using a fluorometer.
100 hundred nanograms DNA on one person agarose gel alongside a DNA molecular weight marker to check DNA quality and quantity. To make circular DNA templates, first digest the DNA with suitable restriction enzyme. Here we use SacI to digest MCF7 and blood genomic DNA Make a digestion reaction mix according to text protocol.
Mix the solution by flicking the tube and centrifuge briefly. Incubate the reaction mix at 37 degrees Celsius for an hour followed by heat inactivation, as per manufacturer's instruction. Next, make a ligation reaction master mix solution according to the text protocol to self-ligate the digested DNA.
For each reaction, add 30 microliters of this ligation reaction master mix directly to 50 microliters of digested DNA solution from the previous step. Mix the solution by flicking the tube and centrifuge the tube briefly. Incubate this reaction mix in a thermal cycler at 22 degrees Celsius for 10 minutes, finishing with a heat inactivation step at 60 degrees Celsius for another 10 minutes.
This circular DNA template will be used in subsequent inverse PCR step. Set an annealing temperature gradient in a thermal cycler to determine the optimal primer annealing temperature by gradient PCR. Prepare a master mix for the PCR by combining and mixing all the components according to the text protocol.
Set up one reaction for each annealing temperature in the gradient. For each reaction, allocate 19 microliters of the master mix PCR tubes and add one microliter of circular DNA template, generated from blood genomic DNA. Run the gradient PCR program.
Run six microliters of PCR product, heat it at different annealing temperature in one course in agarose gel. To detect de novo TTC28 LINE-1 retrotransposition activity in the genome of MCF7 cell line, perform PCR using inverse primer pair on circular DNA templates, generated from MCF-7 cell line. Follow same instructions as for gradient PCR, but this time replacing the temperature gradient with optimal annealing temperature, which is 64 degrees Celsius.
Analyze the long distance inverse PCR products by agarose gel for the results. This agarose gel image taken after a electrophoreses shows the the genomic DNA extracted from MCF7 and blood samples have high molecular weight, making it suitable for long distance inverse PCR. This agarose gel image taken after electrophoreses shows that the inverse primer pair that we designed amplifies the correct circular template at all annealing temperatures in the gradient as observed by intense span between 5 kilobase and seven kilobase DNA marker.
However, various bands are also observed at lower temperatures, indicating poor primer specificity and poor annealing temperatures. Thus, for this primer pair, 64 degrees Celsius is the optimum annealing temperature. This agarose gel image shows the PCR product obtained after performing long distance inverse PCR upon DNA extracted from MCF7 cell line and normal blood.
A PCF product of 5, 700 base pairs, marked by an asterisk, represent the native PCR corresponding to Line-1 at its native locus. This native PCR product is visible in both MCF7 and normal blood DNA. De novo LINE-1 retrotransposition and MCF7 genome is detectable as PCR product of different sizes, along with native PCR product in the agarose gel.
Sequencing the long distance inverse PCR product by single-molecular long-read sequencing technologies can allow identifications of target identification in a nuclear-tight resolution. Although a cumbersome approach, cloning and sanger sequencing of the long distance inverse PCR product is also possible. In this video, the activity of a LINE-1 on chromosome 22 was detected.
The same workflow can be adapted to monitor other LINE-1s that are active in different tumor types.
