Dear Dr. Suhail Ayesh

You may start with blood genomic DNA extraction by using a DNA blood kit followed by quantification using nanodrop and quality check by agarose gel electrophoresis.

This could be followed by a simple method namely, PCR-RFLP. In this method, known regions of DNA are amplified by PCR, and the PCR amplicons are then digested with restriction enzymes to produce distinct polymorphic fragments which are then subjected to electrophoresis. Ultimately, what you detect is nucleotide variation for a restriction enzyme recognition site. The variation must lie within the site.

So, PCR-RFLP employs four main steps:

(1) isolation of genetic material and PCR,

(2) restriction digestion of amplicons,

(3) electrophoresis of digested fragments, and

(4) visualization.

PCR-RFLP is a low-cost and low-throughput research method allowing for the analysis of SNPs in the absence of specialized equipment, and it is useful when there is limited budget.

Besides the above, there are many novel and reliable techniques used to assess gene polymorphisms such as Taqman assay, amplification refractory mutation system (PCR-ARMS), high-resolution melting and different types of mini-sequencing, which you could try, if you have enough funds.

Best Wishes,

Malcolm Nobre

Dear Dr. Nobre.

thanks for your answer and help, some of these procdures we are doing in our lab. since we need to do 20 SNPS for each sample, the time for sample analysis is longer than we need. I need some help in finding some methods like: small snp panel chips, that can be used with small , hand held or mobile instruments.

Dear Dr. Suhail Ayesh

If you want the cost effective and straight forward method, following are few approaches to study obesity-related gene polymorphisms.

(1) Sample Collection and Extraction of DNA [Default method]

(2) RFLP Analysis

(3) PCR Amplification

(4) Agarose Gel Electrophoresis

The easiest method is (1) in which basically, we use a simple DNA extraction protocol, such as blood spot method or a saline mouthwash, to obtain genomic DNA.

While the cost-effective method could be (2) and (3).

If your target SNP has a known restriction site, you can use RFLP to detect polymorphisms. After PCR, the product is digested with an appropriate restriction enzyme, then visualize the fragments using gel electrophoresis. This technique is both simple and low-cost for SNP detection in the absence of

specialized equipment, and it is useful when there is limited budget.

While Polymerase Chain Reaction (PCR) amplifies specific regions associated with obesity-related polymorphisms (e.g., FTO, MC4R).

To analyze the PCR products, you can perform agarose gel electrophoresis also. After digesting the PCR products with the appropriate restriction enzyme, load them onto an agarose gel.

Running the samples through the gel will allow you to observe the resulting fragment patterns. These patterns reveal the presence or absence of the restriction sites, enabling you to distinguish between different genotypes.

To reduce time per sample so as to streamline the SNP analysis , I am providing some efficient options to consider:

1. Custom SNP Genotyping Panels: Small SNP genotyping panels, such as Open Array plates or SNP Array Chips, can be customized to target specific obesity-related SNPs. These panels are compatible with compact devices and can help analyse multiple SNPs in a single run, making them ideal for high-throughput needs.

2. Handheld PCR Devices: Devices like the MiniPCR systems are compact and user-friendly, allowing for field use or in small labs. With multiplex PCR, you can amplify several SNP targets in one reaction, reducing the number of individual PCRs needed and speeding up the process.

3. Multiplex PCR Combined with Micro-Gel Platforms: Multiplex PCR enables simultaneous amplification of multiple SNP regions. After amplification, miniaturized gel systems, such as the Agilent Bioanalyzer, can rapidly analyse fragment sizes, assisting with quick genotyping of multiple SNPs in a single analysis.

4. Digital PCR (dPCR) Systems: Portable digital PCR devices, like the QI Acuity system, can offer precise SNP genotyping with minimal post-PCR processing. Though it may require a higher initial investment, dPCR provides high accuracy and efficiency, making it a suitable option for analyzing multiple SNPs in less time.

Hope it helps,

Aadi Dev Sharma

With these minimum requirements you can proceed your work with genomic DNA extraction and follow up with PCR, and continue withSSCP, single strand confirmation polymorphism studies