Yes, you can perform DNA extraction and PCR to identify your microalgae despite contamination, but you'll need primers specific to microalgae to avoid amplifying bacterial or fungal DNA. For general eukaryotic microalgae, using 18S rRNA primers (e.g., Forward: 5'-AACCTGGTTGATCCTGCCAGT-3', Reverse: 5'-TGATCCTTCTGCAGGTTCACCTAC-3') or rbcL primers (e.g., Forward: 5'-ATGTCACCACAAACAGAGACTAAAGC-3', Reverse: 5'-TCGCATGTACCTGCAGTAGC-3'). Ensure high specificity by optimizing PCR conditions (e.g., touchdown PCR) and consider sequencing the products for confirmation. If contamination persists, isolating single algal cells (e.g., via FACS) can help.

To molecularly identify non-axenic microalgae isolates, several methods and markers can be used. Here are the key points and recommended approaches based on the provided reference materials:

1. rbcL Gene Marker

The rbcL gene (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) is a commonly used marker for the molecular identification of microalgae. This gene is highly conserved and can be used to differentiate between different species of microalgae.

• Procedure:DNA Extraction: Extract genomic DNA from the non-axenic microalgae isolates using standard DNA extraction protocols. PCR Amplification: Use specific primers for the rbcL gene to amplify the target region. Sequencing: Sequence the amplified rbcL gene fragments. Data Analysis: Compare the sequenced rbcL gene fragments with reference sequences in databases like GenBank to identify the microalgae species16.

2. 18S rDNA Marker

The 18S rDNA (18S ribosomal RNA gene) is another widely used marker for microalgae identification. It is highly conserved and provides a good resolution for species-level identification.

• Procedure:DNA Extraction: Extract genomic DNA from the microalgae isolates. PCR Amplification: Use primers specific to the 18S rDNA region to amplify the target sequence. Sequencing: Sequence the amplified 18S rDNA fragments. Data Analysis: Compare the sequenced 18S rDNA fragments with reference sequences in databases to identify the microalgae species210.

3. Combination of Markers

For more accurate identification, especially in non-axenic cultures where multiple species may be present, combining multiple molecular markers can be beneficial.

• Procedure:DNA Extraction: Extract genomic DNA from the microalgae isolates. PCR Amplification: Use primers for multiple markers such as rbcL, 18S rDNA, and possibly other markers like ITS (Internal Transcribed Spacer) regions. Sequencing: Sequence the amplified fragments for each marker. Data Analysis: Compare the sequences with reference databases to identify the microalgae species. This multi-marker approach can provide a more comprehensive identification39.

4. Metagenomic Approaches

Metagenomic approaches can be used to identify microalgae diversity directly from environmental samples, including non-axenic cultures.

• Procedure:DNA Extraction: Extract total genomic DNA from the environmental sample containing non-axenic microalgae. Library Preparation: Prepare a DNA library for sequencing. Sequencing: Perform high-throughput sequencing (e.g., Illumina or Nanopore sequencing). Data Analysis: Use bioinformatics tools to analyze the sequencing data and identify the microalgae species present in the sample3.

5. Fluorescence Activated Cell Sorting (FACS)

FACS can be used to sort and isolate individual microalgae cells from a mixed culture, followed by molecular identification.

• Procedure:Cell Sorting: Use FACS to sort individual microalgae cells into separate wells. DNA Extraction: Extract genomic DNA from the sorted cells. PCR Amplification and Sequencing: Amplify and sequence specific markers like rbcL or 18S rDNA. Data Analysis: Compare the sequences with reference databases to identify the microalgae species

Othman Mueen Mohammed

Theja Virajini

Thank you very much for your generous answers, they will certainly help me in my task.