SSR markers | Touchdown PCR

SSR markers, also known as microsatellites, are short, repeating DNA sequences typically found in non-coding regions of the genome. They are characterized by a small motif of 1-6 base pairs that is repeated in tandem, such as (AC)n or (GT)n. The number of repeats varies between individuals, making SSRs useful genetic markers for studying population genetics, phylogenetics, and molecular breeding. PCR amplification of SSR regions using primers that flank the repeats generates fragments that can be separated by gel electrophoresis and used to identify individuals or populations based on their unique SSR profiles. Touchdown PCR is a modified version of PCR (Polymerase Chain Reaction) that is designed to reduce non-specific amplification of DNA during the initial cycles of PCR. In this technique, the annealing temperature is gradually reduced in a series of cycles. During the initial cycles, the annealing temperature is set higher than the calculated melting temperature of the primers, to encourage specific annealing to the target DNA. In the subsequent cycles, the annealing temperature is gradually decreased in small increments, allowing for the annealing of the primers to any secondary sites on the DNA template, which helps to increase the specificity of the reaction. Touchdown PCR can improve the specificity and sensitivity of PCR reactions, and is often used in applications such as genotyping, mutation detection, and cloning. Problem: Why Smeared and multiple bands occurs in polyacrylamide gel electrophoresis while running ssrs product amplified by touch down PCR? “I am carrying out polymorphism studies using ssr markers and running them on 6% polyacrylamide gel using 1x tbe buffer. But I’m facing smearing type of bands and multiple bands in certain samples can anyone kindly answer why this problem is encountering“. I would run the gel at a lower smiling bands could be caused by too much salt in the sample or by being run at too high a to put lower voltage and dilute samples at least 1:10. Your electrophoresis is to “fast“ according to picture. Seems to me that your lanes are overloaded. Or use a gel with larger lanes for each sample. It looks like you have different template DNA concentration/purity. Each sample must be adjusted to get equal PCR product concentration. #SSR #PCR #gelelectrophoresis