CRISPR/Cas9 - Point Mutations Verification - Alzheimer´s Disease

Wrong letter in the genome code? That was potentially fatal until recently.
Point mutations are the cause of many hereditary diseases and are considered risk factors, for example, for Alzheimer's Disease. One of the simplest and cheapest means of detecting point mutations is SNP Pol Polymerase > or, for real time PCR, SNP PolTaq DNA Polymerase > (High Discrimination).

One of the main causes of many hereditary diseases could not be resolved precisely enough by mid-2016, even with the "super tool" CRISPR / Cas9: the point mutation. If the gene scissors CRISPR/Cas9 were used, it caused frequent coincidental insertions, or the omission of entire DNA sequences. These defects, termed "indels," occurred because cutting the DNA duplex activated cellular repair mechanisms, which then caused such defects. However, David Liu and his colleagues from the Harvard University in Cambridge have modified CRISPR / Cas9 such that the gene scissors no longer cut the DNA. This eliminates the problem of unwanted indels. Instead, the gene scissors can now convert one base into another directly within the DNA strand with the help of attached enzymes - and thus correct the point mutations or set them specifically.

The spontaneous deamination of cytosine is one of the main reasons for the conversion of C/G to T/A base pairs, which are responsible for approximately half of all known pathogenic point mutations in humans. Gaudelli et al. described a targeted conversion of A/T base pairs into G/C, which could therefore advance the research and treatment of genetic diseases. (Gaudelli et al., Nature 2017). 

Using this very strategy, CRISPR / Cas9 has been able to correct such a point mutation in the Alzheimer's risk gene APOE4, for the first time as reported in the journal "Nature". In the United States, more than five million people suffer from this deadly disease. The novel CRISPR / Cas9 technology is designed to substantially block any further progression of symptoms. At the University of Madison, in collaboration with bioengineers, nanoparticles are designed to cross the blood-brain barrier and transport the CRISPR / Cas9 protein into the brain.

The example above shows that with CRISPR / Cas9, a tool is available with which an increasing number of gene defects can be repaired, at least in model organisms. But despite the positive news and principal achievements, CRISPR / Cas9 does not guarantee the desired result every time. Therefore, after using CRISPR / Cas9, it is essential to confirm that the desired gene replacement, in the case of a point mutation, the exchange of a single base, has actually taken place. There are many methods for this. One of the simplest and most favorable is to control / verify the mutation using either the Genaxxon SNP Pol > or SNP PolTaq DNA polymerase (for real time PCR) >, depending on the application. Just direct your primer to the assumed point mutation (the point mutation must be at the 3`-end) and the polymerase will recognise its correct nucleotide target with 100% accuracy: If the 3´-end shows the mutation and the primer doesn´t, then there will be no amplification at all.

Many DNA polymerases tolerate mismatched primer-template complexes! The SNP Pol > and SNP PolTaq > DNA polymerases (High Discrimination of single nucleotides) differ up to 100% by means of allele-specific PCR. A positive PCR result will occur only if the primer pairs are perfectly matched, and will reveal which of the two alleles is present. SNP Pol and SNP PolTaq polymerase carry our "consistently high quality" certification..

Allele-specific PCR can be used to quantify the mutation rate in a pool or background of wild-type sequences. The verification of mutation frequencies determined by NGS can also be achieved by means of allele-specific PCR using SNP Pol DNA polymerase. Thus, the SNP Pol DNA polymerase is an excellent tool of choice for human medicine, food technology or plant biotechnology.

The SNP PolTaq DNA polymerase is very suitable for the analysis of liquid biopsy samples. With it, the presence and frequency of cancer mutations can be very well analyzed and quantified. The variant, SNP PolTaq DNA polymerase, has 5'-3 'exonuclease activity and can therefore be used in realtime PCR together with specific primer probes such as Taqman® probes or Molecular beacons.

Applications for SNP Pol DNA polymerase >
- Monitoring, verification and detection of point mutations
- Identification of correct or off-target CRISPR/Cas9 products
- Verification/validation of sequencing results
- Quantification of mutations (e.g. NGS results)
- SNP-detection by allele-specific amplification (ASA) / Allele-specific PCR
- Methylation specific PCRs (MSP) of bisulfite treated DNA (CpG methylation sites)
- HLA genotyping
- Multiplex PCRs
- realtime PCR with hydrolysis probes
- realtime multiplex PCRs

Literature:

- DamID-seq data in C. elegans / Sharma R, Ritler D, Meister P.
- Minisequencing SNP genotyping with SNPase DNA Polymerase / Lovmar L, Fredriksson M, Liljedahl U, Sigurdsson S, Syvänen AC
- Allele specific mismatch selectivity by the HiDi DNA polymerase / 
Drum M, Kranaster R, Ewald C, Blasczyk R, Marx A

 

 
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