PCR design and genome build compatibility

Question

[laith.moushib]

Hi folks

I am new to molecular genetics and I have some technical questions.

We have done WGS (using hg19 genome build) on some patients and I found two interesting variants:

1. KMT2D: chr12_49426905_TTGC_T ; c.11580_11582delGCA ; p.Gln3863del
2. FLG: chr1-152285861_G_A; c.1501C>T; p.Arg501*

When I search NCBI for gene (cDNA) sequence in order to design primers, I do not see the nucleotide sequence in the DNA sequence on NCBI database for the bespoke genes (that is, I do not see GCA in location 11580 to 11582 for KMT2D and I do not see either C in location 1501 for FLG). Is there something wrong I missed? Is the genome build for these genes on NCBI on hg38 that could hinder seeing the exact nucleotide sequence? How to solve this issue?

In addition, when I design primers should I use cDNA or gene sequence containing introns and exons?

Looking forward for your guidance on this issue

Thanks in advance

Laith

[GenoMax]

I do not see the nucleotide sequence in the DNA sequence on NCBI database

and

is the genome build for these genes on NCBI on hg38 that could hinder seeing the exact nucleotide sequence ? How to solve this issue?

You will need to look at hg19: https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001405.25/

It is a bit odd to use hg19 build at this point. It has been superseded by GRCh38 for almost a decade. You should verify that analysis with current genome build.