Protocol for Designing Primers for Paenibacillus dendritiformis C454 Genes

Purpose

This protocol outlines the steps for designing primers for genes in Paenibacillus dendritiformis, focusing on genes within biosynthetic gene clusters (BGCs) and physiological genes involved in various cellular functions.

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Step 1: Getting the Gene Sequences

A. Genes from Biosynthetic Gene Clusters (BGCs)

1. Identification: Obtain sequences of core genes from BGCs using antiSMASH software.
2. Selection: Choose the core gene from each BGC for primer design.

B. Physiological Genes

1. Gene Identification:
   • Identify physiological genes responsible for essential cellular actions (e.g., cell division, movement, DNA replication).
   • Utilize resources like ChatGPT to list known genes in Paenibacillus dendritiformis.
2. Sequence Retrieval:
   • Search for the desired gene on NCBI with the query “Paenibacillus dendritiformis + gene name”.
   • Prefer sequences from strain J2TS7 if strain C454 sequences are not available directly.
3. Sequence Validation:
   • Click on the FASTA sequence for the gene.
   • Select “RUN BLAST” to compare against a database.
   • Choose ‘Whole genome shotgun contigs (wgs)’ with “Paenibacillus dendritiformis str. C454 (taxid: 1131935)” as the organism.
4. Alignment Check:
   • Verify that the query sequence aligns with the C454 strain sequence.
   • If matched, copy the sequence from GenBank or directly from the alignment.
5. Sequence Comparison:
   • If needed, compare sequences using BioEdit software to ensure similarity.

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Step 2: Primer Design

A. Using Primer3 Software

1. Input Sequence: Enter the validated gene sequence into Primer3.
2. Adjust Parameters:
   • For qPCR primers, set the product size to 130-160 bp.
   • Generate five pairs of primer candidates.

B. Primer Evaluation

1. PCR Primer Stats Check:
   • Run each primer through PCR Primer Stats software.
   • Ensure the primer passes the basic criteria. A warning for temperatures above 58°C is acceptable, with a normal range even up to 62°C.
2. Hairpin Formation (Entropy) Test:
   • Use IDT’s OligoAnalyzer to check the ΔG value for hairpin formation.
   • Select primers with the lowest ΔG value, ideally lower than -1.
3. Specificity Test using PrimerBLAST:
   • Run PrimerBLAST with the following settings:
     • Organism: Paenibacillus dendritiformis c454 (taxid 1131935).
     • Database: Custom -> Full genome FASTA from NCBI.
   • Choose primer pairs with a single expected match, ensuring the amplicon length matches expectations and the match is complete.

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Step 3: Primer List

Prepare a list of primers for each gene, formatted as follows:

Serial Number	Gene Name	Gene Type	Forward Primer	Reverse Primer
1	Asparagine synthase	Natural product	TTATTTCTGGGCAGCCTCCA	GCTGGTCTGCTAATTCGTCC
2	Bacitracin synthetase 3	Natural product	GAATGTCGGTTGGAGTACGC	TTCCTCCTCCGTGAGCATTT
3	Isochorismate synthase	Natural product	TCTCGACCAACATAACCGGG	ATAGTACGCCCGATCGAAGG
4	L-ectoine synthase	Natural product	ACAAACATCATGTGGAGGCG	TCTGGCTCTTTCCTCTCAGC
5	dnaA	Physiological - DNA replication	TGGTTCAAAGCCACTCAAGC	TCCACCTGATTGCCCGTAAT
6	dnaG	Physiological - DNA replication	GGCATCTTGGTGAACGGTTT	TATGGAGGCAGCGACTTTCT
7	dnaN	Physiological - DNA replication	TCCAGACCTTTCTCCGTTCC	GTGGAAATGGCAAAGACCGT
8	gyrA	Physiological - DNA replication	CTATGACGGGGAAGAGACGG	CGCCTGAATGCCATCAATGA
9	gyrB	Physiological - DNA replication	TTGAAGTCAGCTCTCTCCCG	AGATTTTCCCCTTGAGCGGA
10	ftsA	Physiological - cell division	AGCTACTTCGACCTTGCCAA	GCGGACGACTTTGAACACTT
11	ftsZ	Physiological - cell division	GGTAAATACGGATGCGCAGG	AGTTCACGGGACTCTTCAGC
12	ftsI	Physiological - cell division	CGGAATCCTGCAATGTCGTC	GGCATAACGGGTTGAAGCAT
13	minC	Physiological - cell division	GCTTGACGATCAGTGCGAAT	TCTGTCTTTTGCTCATCGGC
14	minD	Physiological - cell division	CGTCAAGGATAAGCGGTTCG	CGGCAGGGCAGTCGATAATA
15	motA	Physiological - movement	GATGACTTCCTTCGCAACGG	CCTGGGAAAAGATAAGCGCG
16	motB	Physiological - movement	GCGGGACAAGATGAACGAAT	GACTTGAGGCTTGCTTTCCC
17	mreB	Physiological - movement	ACGCTGTCCATCTCTTCGAA	GTGAGAACAATGCCCCGATC

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Conclusion

This protocol provides a detailed guide for designing and validating primers for various genes in Paenibacillus dendritiformis. Following these steps ensures accurate and efficient primer design for further genetic and functional studies.