Verifying Primers For Point Mutation Using Primers To Amplify DNA Region Containing Mutation Site

Analysing DNA sequences for determining mutation point in PCR needs paid, Hi-tech software tools but we can confidently order the right piece of primer by this simple and easy method before we do PCR in lab. For instance, we here take BRAF V600E mutation. This method is to verify that the primers you’ve got or designed would successfully amplify the same region of DNA you want and not somewhere else.

For each mutation to be tested, specific sequences are located for primers that allow amplification of a certain target sequence also termed "segment", while the primers for the first PCR harbour is a desired tested location [1]. For example, the amplicon size is substantially 200-400 bp long, melting temperature (Tm) of primers is 63-65° C and primer length is 18-26 bp. The mechanism of mutation is that the negative charge of the acidic glutamic acid residue causes it to be phosphomimetic [2]. The BRAF V 600E mutation includes melanoma [3], hairy cell leukemia [4], papillary thyroid carcinoma [5], colorectal cancer [6], non-small-cell lung cancer [7], Langerhans cell histiocytosis [8], Erdheim-chester disease [9] and ameloblastoma [10].

PROCEDURAL STEPS :
For this method, I have taken a already designed forward and reverse primer from a patented paper [11] where they construct DNA for sequencing, which is an example here for specific primers to the BRAF mutation at amino acid position V 600. The valine (V) will be substituted by glutamic acid (E). So, I'll use that as an example for explaining my procedural steps. We'll see how the DNA sequence codes so primers amplify the right bits of DNA and don't bind somewhere other than that we want.
Dividing the method in two segments, first for identification of PCR product sequence and second to verify that DNA sequence present includes codon 600.
Starting method by listing the primers, I've taken : • Google NCBI website (https://www.ncbi.nlm.nih.gov ). [12] • On the web page we can see " popular resources" on the right hand side.
• On BLAST, we scroll down to "Nucleotide BLAST", click. • There, in "Enter Query Sequence" we type our primer sequence. We will first take the forward primer, copy it and paste there. • Without changing any default settings, Scroll down and click on "BLAST". • It will now take a minute to search all known nucleotide sequences.
• The result of BLAST for forward primer is shown to "descriptions" see top of some human links. (Refer Image :1). • Taking another tab we can follow the same steps mentioned above by taking the reverse primer sequence. Both forward and reverse primer will bind somewhere near Each Other a few hundred base pair apart because that's where the primers are and they should bind to some piece of DNA. Once we know where the forward primer binds and where the reverse primer bind then that allows us to calculate how much DNA there is in between those two primers and it will also give us the total size of our PCR product.
• We will select those links which have mentioned "human" in common name tag list and "100%" in query cover. • Comparing this link of description on both the forward and reverse primer resultant tab, select the sequence that are available to both tabs and shows BRAF • Follow it's "accession", click on it.
Accession are just large genomic fragment of chromosome which happens to be where the BRAF gene is. • We will get a screen showing its details. Scrolling down we find long sequence, our job is to look at where within the primer is in that sequence. Click on FASTA given below NCBI reference sequence ID. FASTA will remove the gaps and numbers. (Refer Image:2) • Which find in page or Control + F we will find the forward primer, type the first half of the forward primer sequence and check for the rest. • Select 5 lines up and 5 lines down from the sequence found. Copy them and paste I Notepad or MS Word. Highlight this forward primer sequence with yellow. • To locate the reverse primer, we need to decode it. As, reverse primer is in reserve antisense. So, we will type it "backwards" and "antisense". Hence, from the selected region of Forward primer to that of the region of reverse primer is the number of base pair in the PCR product we want to use. Checking word count of the selected region will tell us how long our PCR product is. Now, in this DNA sequence, it might be exons or introns, to know this here's SEGMENT 2. Part 2: Find • Search for "ORF Finder" [13] or we can also use "EMBOSS SIXPACK" [14]. ORF finder is basically a open reading frame finder website. • We will copy paste our NM number and without changing any default settings, click on "Submit"

OR
We can copy paste the sequence by clicking on the NM number and run it in fasta format. Copy the sequence and paste it here in the ORF finder "enter query sequence" region. • It will take a minute here, what it is doing is to scan that DNA sequence and look the possible open reading frame, which are basically regions of DNA which convert to copy mrna and translate them to longest runs of amino acids. • Sequence page will appear. Here, in the right side we see column if "length (nt/aa)", we will select the longest one as we need BRAF gene of at least 600 amino acids. Here, it's the amino acid 600 that is changed there's oncogenic mutation to BRAF gene. • We will now click on "Display ORF as" and select "CDS translation". It puts what amino acid codes for particular DNA.