cDNA Synthesis Protocol by Reverse Transcription | Construction of recombinant cDNA from mRNA скачать в хорошем качестве

cDNA Synthesis Protocol by Reverse Transcription | Construction of recombinant cDNA from mRNA

this lacture us helpful for bsc biotechnology ,bsc botany , msc biotechnology,msc biotechnology student and also important for bsc agriculture, molecular biochemistry,molecular biology, molecular biotechnology and msc agriculture student. Hii my name is Nitin and I am life science student.... please support my chennal... In this lacture I am discribe the following point - 1.what is cDNA 2.Method of formation of cDNA 3.isolation of mRNA 4.Synthesis of first strant of cDNA 5.Synthesis of second strand of cDNA 6.Some strategies of formation of second strant of cDNA a. ds cDNA synthesis by self priming method b. ds cDNA synthesis by RNA replacement and Nick translation c. ds cDNA synthesis by random primer e. ds cDNA synthesis by tailing method cDNA (complementary DNA) is synthesized from an RNA template using the enzyme reverse transcriptase, a process called reverse transcription. This step is essential in gene expression studies, especially when using techniques like quantitative PCR (qPCR) to measure the levels of specific mRNAs. In the process, primers are needed to initiate the synthesis of cDNA: 1. **Oligo(dT) Primers**: These primers bind to the poly-A tail at the 3' end of eukaryotic mRNA, ensuring that the cDNA synthesized is specific to mRNA with poly-A tails. This approach is typically used when the goal is to reverse transcribe all mRNA in a sample. 2. **Random Hexamers**: These are short, random sequences of six nucleotides that can anneal to any part of the RNA. This approach is less specific than using oligo(dT) primers, as it can prime the synthesis of cDNA from all types of RNA, not just mRNA. This can be useful when you want to ensure more complete representation of all RNA sequences, including those without poly-A tails or when working with degraded RNA samples. After cDNA is synthesized, it can be used as a template in qPCR to quantify gene expression by amplifying specific DNA sequences and measuring the amount of PCR product in real-time. This allows researchers to determine the expression levels of genes of interest.