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Preimplantation Genetic Testing for Aneuploidies: Where We Are and Where We're Going
Published in Darren K. Griffin, Gary L. Harton, Preimplantation Genetic Testing, 2020
Andrea Victor, Cagri Ogur, Alan Thornhill, Darren K. Griffin
Also loosely falling into the PCR-based WGA category are linker-adapter PCR (LA-PCR) methodologies. In LA-PCR, template DNA is fragmented and tagmented, meaning linkers containing a primer binding site are enzymatically attached to the ends of the fragmented DNA. PCR is then used to amplify the tagmented DNA fragments by adding PCR primers complementary to the linker-primer binder site. While this method can be more labor intensive than other amplification methods, it appears to offer reasonable reproducibility and genome uniformity, allowing for accurate chromosome copy number assessment. This is leveraged in various downstream genome-wide workflows, as various commercially available LA-PCR kits, like Sureplex and Picoplex, are currently some of the most widely used WGA chemistries for PGT-A purposes.
Genetic and genomic investigations
Published in Angus Clarke, Alex Murray, Julian Sampson, Harper's Practical Genetic Counselling, 2019
Multiplex ligation-dependent probe amplification (MLPA) is an application of the polymerase chain reaction (PCR) in which it is not the patient's DNA that is amplified by PCR but, instead, carefully designed probe sequences. Two DNA probes are designed to recognise and anneal to immediately adjacent sequences in a gene of interest; once in place, they are ligated together by a DNA ligase. The two probes carry an additional sequence, not complementary to human DNA sequences, including PCR primer binding sites and additional ‘stuffer sequences’ designed so that the PCR product is of a very specific size. The PCR reaction of the probe sequence, not human sequence, can then proceed, yielding a quantity of product that relates directly to the number of copies of the target DNA in the patient's genome (Figure 5.1).
Next-Generation Sequencing (NGS) for Companion Diagnostics (CDx) and Precision Medicine
Published in Il-Jin Kim, Companion Diagnostics (CDx) in Precision Medicine, 2019
Il-Jin Kim, Mendez Pedro, David Jablons
Another potential caveat for amplicon-based multiplex PCR methods is a complicating multiplex PCR design. Compared to a bait-based hybridization capturing method, GC or AT contents of the primer-binding or amplification regions significantly affect efficient amplification. Germline polymorphisms or mutations at the primer binding sites can cause a failure of amplification or a strand bias.
CRISPR Cas9 based genome editing in inherited retinal dystrophies
Published in Ophthalmic Genetics, 2021
Mayank Bansal, Sundaram Acharya, Saumya Sharma, Rhythm Phutela, Riya Rauthan, Souvik Maiti, Debojyoti Chakraborty
Most recent addition to the CRISPR armamentarium, and modifications is the CRISPR prime editing (15). This technique involves the use of Cas9 enzyme with nickase property (nCas9), a reverse transcriptase, a prime editor guide RNA (pegRNA) with primer binding site (PBS) RNA and a reverse transcriptase (RT) template RNA. Once the target DNA has been identified by the nCas9 enzyme, the pegRNA binds to the target strand, Cas9 is activated and causes a single stranded cut at the non-target DNA strand; the PBS guide RNA binds to the DNA on non-target strand; the reverse transcriptase then forms a desired copy of the DNA on the cut strand, using the reverse transcriptase template. Thereafter, the DNA equilibration and ligase activity happens. The technique, therefore, helps in modifying the genome without the need for potentially deleterious double-strand break generation. Prime editing holds the potential to edit almost all types of mutations; however its use in therapeutic editing, and therefore correction, of retinal dystrophy needs to be proven.
Common light chain chickens produce human antibodies of high affinity and broad epitope coverage for the engineering of bispecifics
Published in mAbs, 2021
Kathryn H. Ching, Kimberley Berg, Kevin Reynolds, Darlene Pedersen, Alba Macias, Yasmina N. Abdiche, William D. Harriman, Philip A. Leighton
Constructs: The common light chain construct CmLC1 was built with a human germline VK3-15/JK1 gene in pre-rearranged configuration. Six pseudogenes with the same sequence, in reverse orientation to the functional V, were inserted upstream, with ~100 base pair spacers from the chicken pseudogene locus between each human pseudogene pair. A unique primer binding site for sequencing of the final construct was included in each spacer region. The chicken light chain promoter (the 2.4 kilobase pair region between the chicken VL and the first upstream pseudogene), J-C intron, and chicken CL were included, so that all of the regulatory elements and untranslated transcript sequences were chicken. An attB site was included for insertion into an attP site previously targeted to the IgL locus in chicken primordial germ cells.42 PGC line 229–92 was transfected with CmLC1, producing ~20 single clones per transfection of 5 × 106 cells.
Innovative application of nested PCR for detection of Porphyromonas gingivalis in human highly calcified atherothrombotic plaques
Published in Journal of Oral Microbiology, 2020
Adrian Brun, Hélène Rangé, Bastien Prouvost, Mikael Mazighi, Yvonne Kapila, Philippe Bouchard, Jean-Baptiste Michel
Nested PCR is a modification of the conventional PCR method, which consists of a succession of two PCRs, the second PCR uses the product of the first PCR as sample. Indeed, two sets of primers are used in the nested PCR protocol. The first set of primers (first PCR) amplifies large fragments by binding outside of the target DNA. The second set of primers (second PCR) binds specifically at the target DNA. It is intended to reduce non-specific binding by reducing the amplification of unexpected primer binding sites. Different methods have been described for purifying the PCR products from the first universal PCR (using 16S rRNA gene universal primers). One option is the Denaturing Gradient Gel Electrophoresis (DGGE) technique, which involves using a gel extraction kit after the amplified products have migrated on an agarose gel. Alternative options include using PCR purification kits or incubation with an enzyme that removes unincorporated primers and dNTPs. Use of the Illustra ExoProStar® (Dutcher) product for removing unincorporated primers and dNTPs is easier to implement compared to other methods, and was highly effective in our study.