Bisulfite Sequencing Primer Design - (Jun/19/2005 )

I am doing bisulphite sequencing currently. However, after the bisulphite treatment and the purfication of dna, i tried to amplify the dna, but i couldnt. i wonder if i have problems in the primer design.

here i use two genes as examples:

NUCLEOTIDE: NW_047338.1

Location/Qualifiers
source 1..2536525
/organism="Rattus norvegicus"
/mol_type="genomic DNA"
/strain="BN/SsNHsdMCW"
/db_xref="taxon:10116"
/chromosome="10"

E.g 1 gene complement(47503..91067)
/gene="Imp1"
/note="Derived by automated computational analysis using
gene prediction method: BestRefseq. Supporting evidence
includes similarity to: 1 mRNA"
/db_xref="GeneID:303477"
/db_xref="RGD:708580"
cpg island, for example, from 90000-90200

E.g. 2 gene 109124..117220
/gene="Gip"
/note="Derived by automated computational analysis using
gene prediction method: BestRefseq. Supporting evidence
includes similarity to: 1 mRNA"
/db_xref="GeneID:25040"
/db_xref="RGD:2709"
cpg island, for example, from 110000-110200

i am not really sure the differences of designing bisulphite sequencing for these two genes. could you use the examples to design the primers to show me the differences, step by step? THank you so much.

Samantha

Hi Samantha,

have you tried some of the tools found in the following link?

Online Tools for Study

I would suggest you have a go with PerlPrimer, people have also been using methprimer but I am a bit suspicious about how it goes about designing primers.

Also please check out Warnecke et al 2002 Methods 27(2):101-7, this paper gives some good tips on primer design.

Nick

Thank you so much for your reply, nick. However, my biggest problem is i am not very familiar with how to read the information in pubmed. I just wonder if i should reverse the sequence to its complementary to design the primers in example 1, but i dont have to do so in example 2.

I could be wrong about the exact definition of it but with your example 1, the annotation is the complement of genomic contig (ie: pointing right to left, and on the complementary strand) the seqeunce given within the annotation is read 5' to 3' and of the complementary strand so there is no need to manipulate this.

CpG islands are usually found at the 5' end of genes if they regulate that particular gene, you do find islands residing in the 3' end of genes although their role and significance remains to be clarified. You should obtain at least 1kb up and downstream of your gene of interest to locate a possible CpG island to study for those genes.

good luck

Nick