Lab Protocol: Primer Design and Phylogenetic Tree Construction

Introduction

This protocol outlines the steps to design primers and construct a phylogenetic tree. We will use commonly targeted genetic regions, retrieve sequences, perform sequence alignment, identify conserved regions for primer design, and ensure primer specificity. Finally, we will construct a phylogenetic tree using MEGA software.

Materials and Tools

• GenBank or similar database for sequence retrieval
• ClustalW or MUSCLE for sequence alignment
• Primer3 or Primer-BLAST for primer design
• BLASTn for primer specificity check
• MEGA software for phylogenetic tree construction

Step 1: Identify a Suitable Genetic Region for Amplification

Common targets for algal species identification include:

• Ribosomal DNA regions (e.g., 18S rRNA, ITS1, ITS2)
• Chloroplast genes (e.g., rbcL, tufA)
• Mitochondrial genes (e.g., COX)

Select the target sequence according to your purpose.

in my research proposal i want to study the affects of clement chang on Fucus vesiculosus, so my Genetic Region of inters will be its rbcL gene

Step 2: Retrieve Sequences

1. Obtain sequences of the chosen region from a database like NCBI GenBank. search in the Nucleotide option

   >Fucus vesiculosus rbcL

   ATGCCTGAGAACGTACAGGAAAGAACTCGATTAAAAAGTGAGCGTTATGAATCTGGTGTAATCCCATATG CTAAAATGGGATATTGGGATGCAGATTACAACGTTAAAGATACTGATATTCTAGCTCTATTTCGTATAAC TCCACAACCAGGTGTAGATCCTGTGGAAGCTGCTGCTGCTGTTGCTGGTGAATCTTCAACTGCAACGTGG ACAGTAGTATGGACAGATTTACTAACTGCTTGTGACATCTACCGAGCGAAAGCATATCGTGTGGATCCTG TTCCAGGAACAAATGATCAATATTTTGCATACATTGCTTATGAATGTGATTTATTTGAAGAAGGTTCTCT TGCTAACTTAACCGCATCTATCATTGGTAATGTATTTGGTTTTAAAGCTGTTAAAGCATTACGTTTAGAA GATATGAGGATTCCTTACGCTTACCTAAAAACTTTCCAGGGTCCTGCTACAGGTGTAATTGTAGAACGAG AAAGATTAGATAAATTTGGACGTCCTTTATTAGGAGCTACTGTAAAACCTAAATTAGGTCTTTCAGGTAA AAACTATGGGCGTGTTGTTTATGAAGGTTTAACAGGTGGTCTTGATTTTCTTAAAGATGATGAAAATATT AATTCACAACCTTTCATGCGTTGGAAAGAACGTTTCTTATATTGTATGGAAGGTGTTAACCGTGCTGCTG CTGCAACTGGTGAAGTTAAAGGTTCTTATCTTAATGTTACCGCAGCAACAATGGAAAATATGTATGAACG TGCTGAATACTCTCATGCTATCGGTAGTGTAATTTGTATGATTGATTTAGTTGTTGGTTATACAGCAATT CAAAGTATGGCTATTTGGGCACGAAAAGCTGAAATGATTTTGCATTTACATCGTGCAGGAAATTCTACAT ATGCCCGTCAAAAAAACCATGGTATTAATTTCCGAGTTATTTGTAAATGGATGCGTATGTGTGGTGTAGA TCATATTCATGCTGGTACAGTTGTTGGAAAATTAGAAGGTGATCCTTTAATGGTTAAAGGTTTCTATAAC ACACTATTATTAACAGAGCTAAAAATTAATTTAGCAGAAGGTTTGTTCTTCGATATGGATTGGGCATCTC TTAGAAAATGTGTTCCTGTAGCTTCTGGTGGTATTCATTGTGGTCAAATGCATCAACTTCTTTACTATTT AGGTGATGATGTAGTTCTACAATTTGGTGGTGGTACAATCGGTCACCCTGATGGTATACAAGCAGGCGCT ACAGCGAATCGTGTTGCGTTAGAAGCTATGGTTTTAGCTCGTAATGAAGGTCGTGATTATGTTGGTGAAG GTCCTGAAATTTTACGTACAGCTGCTAGTACTTGTGGACCATTAAAAGCAGCTTTAGATTTATGGAAAGA TATTACTTTTGAATATACTTCAACAGATACACCTGATTTCACTGAAGTGGCAACTGAAAGTAACTAA

2. Retrieve sequences for several species that are closely related to the target species.

   we will retrieve sequences from the four most abundant seaweed species In various North Sea aquatic environments: Ulva lactuca, Saccharina latissima, Laminaria digitata, Palmaria palmata and Fucus serratus

   >Ulva lactuc arbcL

   GCTGGTGTAAAAGATTCCCGTTTAACTTATTACACGCCTGATTATCAAGTAAAAGATACTGATATTTTAG CAGCGTTTCGTATGACTCCTCAACCAGGAGTACCAGCAGAAGAAGCAGGTGCAGCTGTTGCTGCTGAATC ATCAACAGGTACTTGGACAACTGTATGGACTGATGGTTTAACATCGTTAGATCGTTATAAAGGTCGTTGT TACGATATTGAACCATTAGGAGAAGACGATCAATATATCGCTTATATTGCTTATCCTTTAGACTTATTTG AAGAAGGATCAGTTACAAACTTATTTACTTCAATTGTAGGTAACGTTTTTGGTTTTAAAGCTTTACGTGC TTTACGTTTAGAAGATTTACGTATTCCACCAGCTTATGTTAAAACATTCCAAGGTCCACCTCATGGTATC CAAGTTGAACGTGATAAATTAAACAAATACGGTCGTGGTTTATTAGGTTGTACAATTAAACCAAAATTAG GTCTTTCAGCTAAAAACTATGGACGTGCTGTTTATGAATGTTTACGAGGCGGTCTTGATTTTACAAAAGA TGATGAAAACGTAAACTCACAACCTTTCATGCGTTGGCGTGATCGTTTCTTATTTGTTGCTGAAGCAATT TATAAATCTCAATCTGAAACTGGTGAGGTGAAAGGACATTACTTAAATGCAACAGCAGGTACATGTGAAG AAATGATGGAACGTGGTCAATTTGCTAAAGATTTAGGTGTTCCAATTGTTATGCATGACTATATTACTGG TGGTTTTACAGCTAACACTTCATTATCTCGTTTCTGTCGTGCTAGTGGATTATTATTACATATTCACCGT GCTATGCACGCTGTTATTGATCGTCAACGTAATCACGGTATTCACTTCCGAGTATTAGCGAAAATTTTAC GTATGTCAGGTGGTGACCACTTACACTCAGGAACAGTAGTAGGTAAATTAGAAGGTGAACGTGAAATTAC TTTAGGTTTCGTTGACTTAATGCGTGATGACTACATTGAAAAAGATCGTAGTCGTGGTATTTACTTTACT CAAGATTGGGTTAGTTTACCTGGTACAATGCCTGTAGCGTCAGGTGGTATTCACGTGTGGCACATGCCTG CATTAGTTGAAATCTTTGGTGATGATGCATGTTTACAATTCGGTGGTGGTACATTAGGACACCCTTGGGG TAACGCTCCAGGAGCCGCTGCAAACCGTGTAGCTTTAGAAGCTTGTACACAAGCCCGAAACGAAGGGCGT GATTTAGCGTCTGAAGGCGGTGATGTAATTCGTGCTGCTT

   >Saccharina latissima rbcL

   TGGTGTAATTCCGTATGCCAAAATGGGATATTGGGATGCTGATTATAACGTTAAAGACACTGATATTCTA GCTCTATTCCGTATAACTCCACAACCAGGCGTAGATCCCGTAGAAGCTGCCGCTGCTGTTGCGGGTGAAT CTTCTACTGCAACATGGACGGTAGTTTGGACAGACTTATTAACTGCTTGCGATATCTATAGAGCAAAAGC ATATAGAGTAGATCCAGTACCTGGAACAAGCGACCAATACTTTGCATATGTTGCTTATGAATGTGATTTA TTTGAGGAAGGTTCTCTTGCAAACTTAACAGCTTCTATTATTGGTAACGTTTTCGGTTTTAAAGCCGTTA AAGCTTTACGTCTAGAAGACATGAGAATTCCTTTTGCTTACTTAAAAACTTTCCAAGGTCCAGCTACTGG TGTCGTTGTGGAAAGAGAAAGATTAGACAAATTTGGTCGTCCTTTCTTAGGTGCTACTGTAAAACCTAAA TTAGGTCTTTCAGGTAAAAACTACGGACGTGTAGTTTATGAAGGTTTAACTGGTGGTCTTGATTTCCTTA AGGATGATGAGAACATTAACTCACAACCATTCATGCGTTGGAAAGAACGTTTCTTATACTGTATGGAAGG TGTTAACCGTGCCGCTGCTGCAACAGGTGAAGTTAAAGGTTCTTACCTTAACATTACTGCTGCAACAATG GAACAAATGTATGAACGTGCAGAGTACGCTCATTCAATTGGTACTGTTATTGTAATGATCGATTTAGTTA TCGGTTATACTGCTATTCAAACTATGGCTATCTGGGCACGAAAAGCTGAAATGATTTTACATTTACATCG TGCAGGTAATTCTACTTATGCTCGTCAAAAAAACCATGGTATTAACTTCCGAGTTATCTGTAAATGGATG CGTATGTGTGGTGTAGACCATATCCATGCTGGTACAGTTGTTGGTAAATTAGAAGGAGATCCTTTAATGG TTAAAGGATTCTACAACACTTTATTATTAACTCAACTTAAAATCAATTTACCTGAAGGTTTATTCTTCGA CATGGATTGGGCATCTTTAAGAAAATGTGTTCCAGTAGCTTCTGGTGGAATCCATTGTGGTCAAATGCAC CAACTTCTTTACTATTTAGGTGACGATGTGGTTCTACA

   >Laminaria digitata rbcL

   ATGCCTGAGAATGTACAGGAAAGAACTCGGTTAAAAAGTGAGCGTTACGAATCAGGTGTAATTCCGTATG CCAAAATGGGATACTGGGATGCTGATTATAACGTTAAAGACACTGATATTCTAGCTCTATTCCGTATAAC TCCACAACCAGGCGTAGATCCCGTAGAAGCTGCCGCTGCTGTTGCGGGTGAATCTTCTACTGCAACATGG ACGGTAGTTTGGACAGACTTATTAACTGCTTGTGATATCTACAGAGCAAAAGCATATAGAGTAGATCCAG TACCTGGAACAAGCGACCAATTCTTTGCATATGTCGCTTATGAATGTGATTTATTTGAGGAAGGTTCTCT TGCGAACTTAACAGCCTCTATTATTGGTAACGTTTTCGGTTTTAAAGCTGTTAAAGCTTTACGTCTAGAA GACATGAGAATTCCTTTTGCTTACTTAAAAACTTTCCAAGGCCCAGCAACTGGTGTTATTGTGGAAAGAG AAAGATTAGACAAATTTGGTCGTCCTTTCTTAGGTGCTACTGTAAAACCTAAATTAGGTCTTTCAGGTAA AAACTACGGACGTGTAGTTTATGAAGGGTTATGTGGTGGTCTTGATTTCCTTAAGGATGATGAGAACATT AACTCACAACCATTCATGCGTTGGAAAGAACGTTTCTTATACTGTATGGAAGGTGTTAACCGTGCCGCTG CTGCAACAGGTGAAGTTAAAGGTTCTTACCTTAACGTTACTGCTGCAACAATGGAACAAATCTATGAACG TGCAGAGTACGCTTATTCAATTGGTACCGTTATTGTAATGGTCGATTTAGTTATCGGTTATACTGCTATT CAAACTATGGCTATCTGGGCAAGAAAAGCTGATATGATTTTACATTTACATCGTGCAGGTAATTCTACTT ATGCTCGTCAAAAAAACCATGGTATCAACTTCCGAGTTATCTGTAAATGGATGCGTATGTGTGGTGTAGA CCATATCCATGCTGGTACAGTTGTTGGTAAATTAGAAGGAGATCCTTTAATGGTTAAAGGATTCTACAAT ACTTTATTATTAACTCAACTTAAAATTAATCTACCTGAAGGTTTATTCTTCGACATGGATTGGGCATCTC TTAGAAAATGTGTTCCAGTAGCTTCTGGTGGAATCCATTGTGGTCAAATGCACCAACTTCTTTACTATTT AGGTGACGATGTGGTTCTACAATTTGGTGGTGGTACTATTGGTCACCCTGATGGTATTCAATCCGGTGCG ACAGCAAACCGTGTTGCTCTAGAATCTATGGTTCTAGCTCGTAATGAAGGTCGTGACTACGTTGGAGAAG GTCCTGAAATCTTACGTAAGGCAGCATCTACTTGTGGTCCTTTAAAAGCAGCTTTAGATTTATGGAAAGA TATTACTTTCGATTACACTTCAACAGATACACCTGATTTCGTTGAAGTTGCAACTGAAAGCAAATAG

   >Palmaria palmata rbcL

   GGTATAAGTTAGTCTTTATATGATTATCAACTATTAGTCTTTAAAGACAATTAAGATAGAAACCTAAGAA ACTGCCTTAAATTCAAGGAGGAATACATGTCTCAATCCGTAGAACAACGGACAAGGATCAAAAACGAACG TTATGAGTCTGGAGTAATTCCATATGCTAAAATGGGATATTGGGATCCTGAACACGTAATTTTAGAAACT GATGTATTAGCTTTATTTCGTGTTACACCTCAACCAGGTGTAGATCCAATCGAAGCTTCTGCTGCAATTG CTGGTGAATCTTCAACAGCAACTTGGACCGTTGTATGGACAGATCTATTAACAGCTTGCGACTTATACAG AGCAAAATGTTATAAAGTAGATGCAGTACCAAATACAAGTGATCAGTACTTCGCATACATTGCATATGAT ATTGATTTATTTGAAGAAGGATCAATTGCAAACTTAACTGCTTCAATTATTGGTAACGTATTCGGATTTA AAGCTGTAAAAGCTTTACGTCTTGAAGACATGCGTTTACCAGTTGCATACCTTAAAACTTTCCAAGGTCC TGCAACAGGAACAGTTGTAGAACGTGAACGTATGGATAAGTTTGGTCGTCCTTTTCTAGGTGCAACTGTA AAGCCTAAGCTAGGTCTATCTGGTAAGAACTACGGGCGAGTAGTGTATGAAGGTCTAAAAGGTGGTCTGG ACTTCCTTAAAGATGATGAAAATATTAATTCTCAACCATTCATGCGTTATAGCGAAAGATACCTTTACTC TATGGAAGGTGTTAACCGTGCTCAAGCTGCTGCTGGTGAAATTAAAGGACATTATCTTAATGTAACAGCT GCTACAATGGAAAACATGTACGAAAGAGCTGAGTTTGCTCAACAACTAGGGAGTGTTATTTGCATGATTG ACCTTGTAATTGGTTATTCAGCTATTCAAAGTATGGCAATCTGGGCTCGTAAGACAGATATGATTTTACA TTTACACAGAGCTGGTAATTCAACATACTCTCGTCAAAAAATTCATGGTATGAACTTCCGAGTTATTTGC AAGTGGATGCGTATGGCTGGAGTTGATCATATTCACGCAGGTACTGTAGTTGGTAAATTAGAAGGTGACC CTGTAATGATTCAAGGTTTCTACAATACTTTACTTGAGAGTCATCTTGAAGCGAACTTACCTCAAGGTAT CTTCTTCGAACAAGATTGGGCTTCACTAAGAAAATGTACTCCTGTTGCTTCTGGTGGTATTCATTGTGGA CAAATGCATCAACTGTTAGACTATCTTGGAGACGATGTAGTTCTTCAGTTTGGTGGTGGTACAATTGGTC ACCCTGATGGTATCCAAGCCGGTGCAACTGCAAATCGTGTAGCTTTAGAGAGCATGGTAATTGCAAGAAA TGAAGGACGCGACTTTGTTGCTGAAGGTCCTCAAATTTTGAGAGATGCAGCAAAAACTTGTGGACCTCTA CAAACAGCTTTAGATTTATGGAAAGATATTAGTTTCAACTATACCTCTACTGATACAGCTGATTTCGTTG AGACTCCAACAGCTAACGTTTAAATATAACTAGTCTTAAGTTTACTTCTAATTAAATTAGAAGATTAAAT TTTAGATTACTACTTGCCTAATCATTAAAGGAGTATAGAATAGTGAGATTAACACAAGGAGCTTTTTCCT TCCTGCCAGATTTGACAGATGACCAAATTTCAAAGCAAGTATCATATGCTATGGGAAAAGGTTGGTCTGT AAATATCGAGTATACTGATGATCCACACCCACGTAATTCCTACTGGGAACTGTGGGGTCTTCCACTATTT GATATTCCAGATGCACCTACTGTAATGTATGAAATTTCTAGTTGTCGTAAAGCACATCCAGGAGTGTATA TCAAAGTAAACGCTTTTGATAATACTAGAGGAGTTGAAAGTTGTTCTTTATCTTTCATCATCAATAGACC TGCTTCTGAACCTGGTTTCGGACTTCTACGTACTGAAGATATTGGACGTAACCAAAAGTATGCTATTCAT AGTTATGCTACAGAAAAACCTGAAGGCTCTCGTTACTAGAGCATTAAGTAATATTTATTCATAACTTATG ACTAAAAAAAGAAATTATAGAAATATAATTTCTTTTATTTATTTAATATTAACTTAAAAAATAGTATGCC ACAAAAATTTTCAGACGATACT

Step 3: Align Sequences

1. Use a tool like ClustalW or MUSCLE to align the sequences.

2. This alignment helps identify conserved and variable regions. conserved regions will be markt with *

   see alignment hayer

Step 4: Identify Conserved Regions

1. Look for conserved regions within the alignment flanked (*) by variable regions.

2. These conserved regions will be the sites for primer design.

   

Step 5: Design Primers

1. Use a primer design tool such as Primer3 or Primer-BLAST to design primers in the conserved regions.

   we will use the conserved regions: TATCGTGTGGATCCTGTTCCAGGAACAAATGATCAATATTTTGCATACATTGCTTATGAATGTGATTTATTTGAAGAAGGTTCTCTTGCTAACTTAACCGCATCTATCATTGGTAATGTATTTGGTTTTAAAGCTGTTAAAGCATTACGTTTAGAAGATATGAGGATTCCTTACGCTTACCTAAAAACTTTCCAGGGTCCTGCTACAGGTGTAATTGTAGAACGAGAAAGATTAGATAAA witch is highly conserved in the middle of it and les conserve at the eggs

2. Consider the following criteria:

   • Primer length: 18-25 nucleotides
   • Melting temperature (Tm): 50-60°C
   • GC content: 40-60%
   • Avoid secondary structures (hairpins, dimers)
   • Ensure primers are specific to the target species

   

   the primers fit the criterias, with a GC content around 40%, Tm in the rage of 50-60 and do not have secondary structures

Step 6: Check Primer Specificity

1. Use a tool like BLASTN to check the specificity of the designed primers against the entire database. will enter both primers as FASTA format

   >foreds

   TCTCTTGCTAACTTAACCGCA

   >bakwerds

   GGTGTAATTGTAGAACGAGAAAGA

2. Ensure they do not bind non-target species. the blast results show that the primers aline 100% to are species of interest,

Creating a Phylogenetic Tree Using MEGA

1. Prepare Your Sequence Data
   • Collect and format your sequences in FASTA format.

   see FASTA file

2. Align Sequences in MEGA
   • Open MEGA software.
   • Click on Align > Edit/Build Alignment > Create a New Alignment.
   • Copy and paste your FASTA sequences into the alignment window.
   • Perform alignment using MUSCLE.
   • Save the alignment file.
3. Construct the Phylogenetic Tree
   • Click on Phylogeny > Construct/Test Maximum Likelihood Tree.
   • Load your alignment file.
   • Follow the prompts to set tree construction parameters.
   • Generate the phylogenetic tree.

   

Conclusion

Following this protocol, you can design specific primers for the amplification of target genetic regions in algal species and construct phylogenetic trees to study their relationships. Ensure to validate primer specificity to avoid non-target amplification.

References

• Primer3: http://bioinfo.ut.ee/primer3-0.4.0/
• Primer-BLAST: https://www.ncbi.nlm.nih.gov/tools/primer-blast/
• BLAST: https://blast.ncbi.nlm.nih.gov/Blast.cgi
• MEGA: https://www.megasoftware.net/