1. Retrieval of the Gene Sequence
The gene sequence selected for primer design was the Oryza sativa Indica Group DRO1 gene, which encodes the early auxin response protein. The complete coding sequence (CDS) was retrieved from the NCBI database for the cultivar IR64.
Accession: AB689742.1
Gene: Oryza sativa Indica Group DRO1
Cultivar: IR64
Description: Early auxin response protein, complete CDS
2. DRO1 FASTA Sequence
The complete FASTA sequence of the DRO1 gene was obtained, and specific regions of the sequence were selected for primer design. The highlighted regions represent the sequences considered for designing overlapping primers.
>AB689742.1 Oryza sativa Indica Group Dro1 gene for early auxin response protein, complete cds, cultivar: IR64
GTACCTCTGGGGTAGAGGCTAGATCCATCCACCACTCATGGACCGTCGCAGGGTTATTTCAATTTGATGCCTGAATTTGCTCATGGGAGATCCAAGCTGATATTGATCCCCAAATTCTCTGTGTACCTGTATTTCACTATTAGGTGTAGGGCTATCTCCAGAGTATGCCGGCAGAATATGAGAAGTAGGCGTTGTTATGTCATCCGCGGGCCACGAGGTGATCCGACGTCTATACTATGTAACATGGTGTCTTAGAGCCCTCTCTTTTCTTCTAAACCTGTATAGTAAAACATCTTGTGAGCACCCACAGTTTTACGGTTGCTTTCTCGCCTGAACCCCACATCACAGTACATCGATTACGCCTTCTTTCTCCCGACAGGGCTAGTTGATTATCATCTCTTATCCAGTCTGCACGTACGCAAAGCTAGTGTAGGCAAGGAGACTCTTCCAGTAGTCGTTTGCTAGCGGTTTGCAGGGCAAGCACACGTTGCAGGCCGGCACGGCAGACTTGCGCAAATTGACGACGGTGCGAGCACAAGCTGGGCAAGCTGTCTCTCGTCTCGTCATGGATCTCGCTCGCACGGATGCCGTTGTCCATGGCATGAACATGAGACGGCGATGGCGAGAGGACGTACATGTGGGGATGGATAGATGGAGGAGACTGGACTAATCGATCGAGTGTAGTAGGACTGTACAGTACTGTGGTGGGGGGATCGAACAAGGATAATATGCCGGATGCCGACCTCATGCGCCACATGCCCACATAGCAAGCAGCAGGGTGTGTCGTCGCGGTCCGGTCCGCTGTTTGCAGCTAGGGGGTTACGTTGCTGTCTGGAAGTACAGGTTGCAGGAAAGATCTGGAGAGGAAGCAGTGACAGCAGTAGCTTGGTGTGTTGCGTTATGCAAGGGGGCTTTGGAACTTGGAAGTTGAAAGCTCTCTCTGAAGAAGGATCTTCAGGTTAGTTGCAAAGGACGGAGCATATTCTTCCCGTACTGTGAGATTCGTAATCGACATTCCGACTGTACGCATGTTGTACTACTACAAAATGGAGTATGCGCCTAACAAATTGAGGATCTCTTGGCTAAGGGCCTATGTAGTTCGTGAAAAAAAAAAATTTTAGGTGTCACATCGGATGTTTGACCGGATATCGAAAGGGGTTTTCGGACACGAATGAAAAAACTAATTTCATAACTCGTTTGGAAACCGCGAGATGAATTTTTTTGAGCCTAATTAATCCATCATTAGCAAATATAGGTTACTGTAGCACTTATGACTAATCATGGACTAATTAGACTCAAAAGATTTGTCTCGCGATTTCCCCCCTAACTATACAATTATTTTTTAAATTTATATTTAATGCTCCATGCATGTGTCCAAAGATTCGATATAATGTTTTTTGGAAAAATTTTTTAGAACTAAACTAGGCCTAAGAGGAAGGTGATAAGTATCTATCATTTTTTATTTTTTATTTGTAGTATCATAAAATCGGGAAACTATCAGAATTTAGAAAGTATATACTTTTATATGGAATATAGAATATGTTGTATAAATTAAAGTATATAATATTAATTTTGATAAATTTAATTGGTTTTATGTAAACTTAACCATATTAATTCATATTATTTTGAATCCATCTTTTTATAAATAGTATTTTATAGAATCGTGGGGATCGTAGAATCGGTATACTACCTAAGATTTCGTATGATCGTGTGAAAAGAAAGATCAAAATACTATAAAAAGTAGTAAAAAGTAGGATACTAGATAAAATCAGTATCATTTTGGTTTGGTAGGATCATATAATATCATAAAATTGGAGCATACGCCCCACTGGCCAAACCTAAGATATGTTTAAAGAATGGGGGTAGTATATAACGGAATCCCGTTTTCGAACGGGATGATCCCGAGTAGGTATCAGGTGTGATACCTTTCAAGTATCAGACGATTCTACACACGTTTCAGGTGATACTTGCAAGTATCATGCGATACTTATCAGGTATCATGCGATTTCTACCACGTATGGCATGATACTCGCGAGGTATCATGTGATACTTGTCAAGTATCAGACGATTTCTACCACGTATCGCGTGATACTCGTGAGGTATTATGTGATACATGTCAGGTATCAGTCGATTTCTACCACGTATCGCGTGATACTCGCGAGGTATCATGTGATATGCCTGTTAGGTATCAGACAATTTCTACCATGTATCGCGTGATACTTGCGAGGTATCGGATGATACCTACCAGGTATCAGGTGATTCCTACTAGATATAGCTAGGCATCCCGTTGGGGAGCATCCCGACTCGCGCGCGGCCGGCCTACTGTAGCCGTCCCATGTAGACGAGGACGGCAGGGGACGGCGCGGCGGCGGCGGGGAACGGCGACGGCGGCGGTGGGGGATGGGCGCGGCGGTGGTGGTGGGGGACGACGATGGCGGCGAGGGACGGGCGCGGCGACGGGTGAGCAGGCCTGGTTCGCGTTGAGGGCTTCGGCCGCGTCGCATGGGTGAGAATCATGTTGTGGAGGAGGAGCATCCCGTTGGAGGACAGTATCCCGTCCCCTAGCATTCCCGTTAAAGAATTCGGTCTGTGCTCTTGGCTAACCAAATCTAATACGGGAAATTCTGGTCATAAGGCCTCAGTTAACCGTTGACGGATTGAGCAGAAATATAGCACGAATCCAAGCAAACCTACCCTGCTTTTTTATTAAGATATAGATATATATTTTGATTTCTGTTCGCATACTTTTTAAACCGCTAAAAAGGTACGTTTCGTACGGAAACTTTCTACATAGAAATTGCTCTAAAATATTAACTAAATCAATTTTCCAACTAGTAATAATTAAAACTTAATTAACCATTAGTAACTTTCTTTTTTTTATGCTCTTACTTAATCTTAATCTTTATTAGTTTCAAGCACCACCTTATTTTCATCTTTATCCCTAGCTCGTCTTCCCCAACAGTCACGGGCTAAGTCCCAGCACAGTCGCACTCTCCTCCCATCTTGTGCCTCCGCTCCCTCCACTAGCTTGTAGGTGGCTGGCGACAATGCCTCCAGGCCGCTGAAGGAGAGTGCCTGAAGGAGGAGAACGACAACTACAGTTGTGGGATTGAGGAATTGCAGGCAAAATTTACTCTCAAAACATGTATCGAAATTGATCGGTCAGCTCGGCCATGACCGAAGACCGAACTATAAAAATGATCAAATATTTTAGGTTTTACGGTTTTATATTAAATTTCGATCTTGATTTTAGAGGCTGAAGTTTTGAGAGGACCGAAAAACCAAACAAAATTAATCGGTCAGACTGAATAACCACCCCTACTCACCCACGTAGCGATAAGAATGTGTGTTTAGAAATTGACTAGAGATATCTAATTAGTAGAATTTTCGTTAGCGCTTCTAATCTAAACTGATAAATTTGGTTTTGTTACAATGTATTGCCACAACGTAATTTTAGTTTTGCAGAGATATATATATATATATATATATATATATTAGCACTATCATGTCATGTTAGCTTGTTGGTCCAGGTGCATCCACGGTCTAGTACCATTATTGCGTAGTTAATGGACGGAATAGCTGCTAGTGGAGATGGGGGTGTACTTCAGCAGTTAACCCCCAGAAACGTGATTAGCATCGGTGCAGGCGCCCCTCGTGCTGGTTAACCAAGGCGGGCGCCCCGTACTTACCCGCGCTAATGCCGGGTTTAAGGAAGTTAACACGTGATTCTGGCACACGCGTCGGGCCCGACCAAGTGCGATGGGATCCGTTGCGGCGCGGTCGTGACACTTGGTGCCCCAACTTGCCGTCGATCTCGCCGCGCTGTACTTTATCTTAGCTAATAACTCTTCTTCTCCCCCTGGAAAAAAAAAATTAACAACCGGGTTTCTCCCTTTGAGGCATTGATTTTTGGATACTCTCTCGCTGGCTGCAGTGCAAGCGAACCAGTGCACGGTATTCAAACAGTCTGCAATACCTGGGTAAATCTGAGTCCTTTGGTTTTGATCTGGTGAACATAAGAGCACCCGCAATGGTAAAGTAAGGTCCTATCTATAAAACATGTACATCTCAGCAATAGACTCGATTAATAGTAAACCACCTTAATAGTATGTCTATATTGGTATCTATAGCTCTCTCATGCATTGCCTCGTTTTTCTCTATAGACTATCTCTAAGTTAGTAGATAGTTTTGCTCTCTCTCTTCATTTAATACATTCCAAGTAGGAAAATATGCTGACATGGATCTCTTGTAGAGAGCCTATAGATAACCATTGTGGGTGCCCTAATATGACTGTTTAAAACTACAGTCACCCTACTTATTGCAGCTGAACCGTCAAATTATAGCTTCTTCTAGGGATGGATTGGGAATTTTCTTACATGTTGAGTTTTTAACACCTGACTAGACAATCCCACGATATACTACCTTCGTCCTAAAATATAGTAATTTTTAGCTATGAATTTGGACACACAAAAATTGCTAATATTTTAGGACAGAGGGAGTAGTCAGATAGTTTTATCATTCTTAAGAAGGTATATCGAGATGATGGTTTTTTATCGTAAAACTTAATATCTCTAGTAGGTGTCTCGAAATATCGTACAAGTCCTGCATGTACTTTCTTAAGTACCATAAACTTCTCTACAATTTAAGTAGGGTAGAAGCACCCCCGTTGAGGTGTAGCTACCACAATTTTGGGTTCCTCAAAAGTGTGAGGTCGATCACAAGTTTACACTATCTATATCTAGAAACATCAATTCAATATTTACAATCCGTATGGCGCATATGGGCGTACGGTAGCTGGAGTTGCAAATACCGAATTTTCTCTTCCGAAGAAAGTGAAAATTCCATTGTTTATATATATAGCTGTGTCCTGTTATCATTCCATGGCTCACCTGTTGAGCTGGTAACCCGGTTTGGAGTCATCTATAAAAGGAAGGTAAAGGAAGTGACATGCCAGATGAAGTATAGCAAGCTATCCTACGGCCAGCCACGCTGCACCGAACCCTCCAATCCTTGGAACTTGAACCCCTTCAAATCACACCCAGTAGGCTACTAGTAGTACTTCTCTGCACCAATTCCATCGCCAATAGCAGCCACTACAAGTTTTACATCTCTCCTTTCCTCCTCTCTCTGCCATTGCTAGGAGCTTGCATTTCTTGGTAGCTTCATCAGCTAGCTGCTTTCTCCCTCCCCAATCTCTCATTCTTCAGGCCAGGATATGAAGGTAAAGGCTCAAACCATCGGTGTGATTAATCATTTCAGAGAGGTTAATAGATTTGAAGTGTTGGTAGTGTTGATCCATTTCTTGATGGCATCATGAGGCTTGGGTTTTCTCTGCAGATTTTCAGCTGGGTAGCCAACAAGATCAGTGGGAAGCAAGAAGCAAATCGGTTTCCCGCAAATTCTTCTGCGCCTTACCGTGAGTGTACCTGAACTTTCAGTTTCCTTGTAAATTCTTTCAGAACTAAGGTGATTGTTGTTCACCAGGCTTCATGCTGTTCTTATGAAATGTTTCCCTGTTCCTTGAGGGGAGAGGGAGAGTACGGACAGTAATTTAGAAGCCGTTTTCTAATTTAATATTTTCTTTCTGTCATTCTGCTGCAGTTATAATGTCTTTACCTCTTAGCAGTTTCTGCTTAGGCATCATTTGCCTTGTCCTAATTTAATCTTTTCCTGCTGTCATTCTGCTCCATTTATGGTGTCTTTACCTCTTAGCAGTTTCCGCTTAGGCATCATTTGCCATGTTCATTTCTGAATATGCTGATATACTTTGTTGTCTTGGTAGCCATATACAAGAGTGAACGATTTAAAGGTTTCTCCTAATTGCTAGTAACAGAACGCTTTTCGCTCTGCGATTTTCTGGATTACCATCCCCTGATTCCTGAGTAACCTATCACAACGCCAATGTGATGGAGCAAACATTGATGTATTATGACAATGTTATTTCTGATCCTTTGAGAAAATTTTCTTATATGCACTTGGGTGTTGTTTGGAACGCAGGAATTTCACTGGAAACAGTTCAATTATCGCACTTCCGGAAAAAAAATTCCTGCATTTTAAACGGCGCTTTAAGTTCAGTAGGAGTTTTCAATTTTATTTTCCTCTTCCCTAGCACGAAAAGTGAGAGATTTTTGTGCATATGTTATACCCCCAAAGGACTATTTCATGGCCTGATGCCCGCCACGCCAATATCTCTGTAAAGCACTGCTTCCAACTAGTCAGTATGGGTCAAAGTCTTGACTAAGTGCAACCCTGCAAGCAAGTGTTTCCAACTAGTCAGTATGGCTTCAATTATTTAGTTGATTTATAACCCCAACCCTCCACTTGTGACCAAGAAGAATTTGATTTGAGCAGACAATATCCTGACCTCGGATGGTTTCGTAAATCGTACTAAGGAATCAGATCTGAGCCACTAGTCCAGCTGGACTACAGTACATTATTTATCTCTCAAGATTACATAAAAAGACTACTATTGTACAGAGAATGCATTGTTTAAGTGGTAACAACTGCTGTGATAAGTTATTCCTGGTATACAGAAAAATGGCAGGTGAAAACATCAGGGAGTGGAGTAAGCATGGGCAGACATTGTACTTAAAGAGTGATAGGCCAATGAGAATCACTGGGTGTTTAAGGATTAAAAATTTGTGGTCCATGCTCTAGACATCCAATTTATGCTAATATGAACTTAGGACCATATATTTATGTTTGGTTACTGATCTTGCGGCTTAATCGAGTTCTAATGCGCAGGTGCTAACGTGTCAGATTGTCGAAAGGATGAATTCAGTGATTGGCCCCAATCATTGCTTGCTATTGGGACATTTGGAAATAAGCAGATTGAGGAGGTAGCTCAGGTGGAGAATTCATCCGACAATGTGCAGTCAGTGCAAGATACCGTCAAATTTACAGAGGAAGAAGTAGACAAGATACGGAAAGAATTCGAAACGCTACTAGCAATCAAAGATCAAGCAGAAGCTCAACGCTCGCATGATGATGACCAAGTAGGTTTACAAAAGCGCGCCGATGGGGAAGACAATGAGAAGCATATTAGACAGTTGATCAACAAAAGGATCATTGTAAGTAAATCAAAGAATTCGTTAGGAAAGAAAGGAAATACACTCAAGCCGAGATCAGTCGCTTCGTTGCTCAAGTTATTCATGTGTAAGGGTGGCTTTACCTCCGTAGTTCCAGAACCAAGGAACACATTTCCTCAATCAAGAATGGAGAAGGTACACAGTGAAATTCTTTTTTTCTTATGTAACATGATAAGTTTTACATACTTTCTCGTCCTCTTATTATCTTATATGATGTTCATTGCAGTTGCTCAAGGCAATATTGCAGAAGAAAATACACCCGCAAAATTCTTCCACGCTAGTTGCTAAGAGGCATTTGGACTGGAAGCCAGATGAGACAGAAATCAATGAGTGCCTGGAGGTGCACTGCGTGATCTAGACGATGATGGTGCAAAATGGGTCAAAACTGATTCAGAGTGTAAGTAGCATACTTGCCTATCATGAATTGAACCTTTTCTTTTGCCACTTTTGTCAATGGAGCAACTGACTGAATATGAACTTTTCTTGTTTCCAGATATTGTGCTTGAAATGTAAAGTTGGGTGAATATGTTAGTTCGTTCATGCCAGGTATATACCTTTCTTTGATCCCCAAATTTTCAACTCAACTCAAATGTGAATTATCCTTGATCTAGCATGTCTCTCTGTTTTGATCTGATTAGTGTGATTCCAATCTGCAGGTTCCAAAGGTCCTGGCAAGCTTGGGGTTTATGGTGTTACTACGTAATGATATAAATATGGGATATAGTTAGCAATGAAGCTATGATCATGTAATGCTCCTCCATTATTTCTGACATGAACCATCTGTAATTTGAATCATGATAAGGAGGTTCTGGGACAAAGGCCTATTCCATGTGTCTACTCTCTTCTGCCCTGAAACTGTAAAGAACGCATTCAATTTTTTCAACTATCTTCTATGACAATTGCCATCTGTTGATTCCAGAGTTGTCTGCCATATCCAATTCAATTCACTAGTTAAATTCACATCATCTCTAACACTTGCTGAATAACTGTTTGATCACCTGATTTCTACACTCCAATAAAGAGTAATAAAACGGTAAGATTGACAACGTTGTTGGTACAGTACGGGCAGAGCGTAATATCTTGTCCGTGTGGCAGAGAAGAAAACGCACCTTCCATCCGTTGCAGGCACGGACGCCTGTCTGTGTTGCGTTGCGTTGCTGCTGTACGTTCTTGGGAGCTTGTGCTTGGACACGCTTGACATCGGCGAGATTCTGCCGGCCCGTACCCCGGCCATACGTGATGGCCGGGCAGGATTCCCTCGTCCACGAGAGAGCATGGCGCAACCGCTGCTCCGGTGAACCTCCCCCCTACGTTAGATCTGGCAGAAGAGCGTATTCTCGTGGCAGCTCCATCAACTCTCTCCTTGACTTACCTCAAAAAAAAAAAACTCTCTCCTTGACGCAAATTAAGCTTAGCTCGACTAGTTGGATTACTTGCGGTGGAACCAATTCACCCGGATTGAAATTCTAGAATTGATATATATGTTCGGCTAATTATTTTTTCAATGGTAGGGTAGACGTATCTGTCGATAGCGAGACGCCTATAGTGACTTCGTCCTTAAAATATGTCGACCTAGTTTTCCGAAAGTACTTATATGAGTAGGATGTTTGAGTGTGTACGTGTTACAAGCGTCTGCGTCTGATATGTGTTTCTTAAAAAAAAAAACTCTCCTTGACGAATCTACTGCACTGCAGAAATAATTCGCGTTCGTGCACTCCTCCATCAGACGAGTAGCGGTCCATTGATTGTGCGATTGTCCCAGCCACATAGGTGCGAGAAGGCTTCCCGAGAAGGCCGGCCCAAAAATTCCTTCGCGAAAACGAGCCGATTCCGCTGTGAAATCGGCGGCC
Now we design a primer and we define primer sequence by different text highlights color:
GTACCTCTGGGGTAGAGGCTAGATCCATCCACCACTCATGGACCGTCGCAGGGTTATTTCAATTTGATGCCTGAATTTGCTCATGGGAGATCCAAGCTGATATTGATCCCCAAATTCTCTGTGTACCTGTATTTCACTATTAGGTGTAGGGCTATCTCCAGAGTATGCCGGCAGAATATGAGAAGTAGGCGTTGTTATGTCATCCGCGGGCCACGAGGTGATCCGACGTCTATACTATGTAACATGGTGTCTTAGAGCCCTCTCTTTTCTTCTAAACCTGTATAGTAAAACATCTTGTGAGCACCCACAGTTTTACGGTTGCTTTCTCGCCTGAACCCCACATCACAGTACATCGATTACGCCTTCTTTCTCCCGACAGGGCTAGTTGATTATCATCTCTTATCCAGTCTGCACGTACGCAAAGCTAGTGTAGGCAAGGAGACTCTTCCAGTAGTCGTTTGCTAGCGGTTTGCAGGGCAAGCACACGTTGCAGGCCGGCACGGCAGACTTGCGCAAATTGACGACGGTGCGAGCACAAGCTGGGCAAGCTGTCTCTCGTCTCGTCATGGATCTCGCTCGCACGGATGCCGTTGTCCATGGCATGAACATGAGACGGCGATGGCGAGAGGACGTACATGTGGGGATGGATAGATGGAGGAGACTGGACTAATCGATCGAGTGTAGTAGGACTGTACAGTACTGTGGTGGGGGGATCGAACAAGGATAATATGCCGGATGCCGACCTCATGCGCCACATGCCCACATAGCAAGCAGCAGGGTGTGTCGTCGCGGTCCGGTCCGCTGTTTGCAGCTAGGGGGTTACGTTGCTGTCTGGAAGTACAGGTTGCAGGAAAGATCTGGAGAGGAAGCAGTGACAGCAGTAGCTTGGTGTGTTGCGTTATGCAAGGGGGCTTTGGAACTTGGAAGTTGAAAGCTCTCTCTGAAGAAGGATCTTCAGGTTAGTTGCAAAGGACGGAGCATATTCTTCCCGTACTGTGAGATTCGTAATCGACATTCCGACTGTACGCATGTTGTACTACTACAAAATGGAGTATGCGCCTAACAAATTGAGGATCTCTTGGCTAAGGGCCTATGTAGTTCGTGAAAAAAAAAAATTTTAGGTGTCACATCGGATGTTTGACCGGATATCGAAAGGGGTTTTCGGACACGAATGAAAAAACTAATTTCATAACTCGTTTGGAAACCGCGAGATGAATTTTTTTGAGCCTAATTAATCCATCATTAGCAAATATAGGTTACTGTAGCACTTATGACTAATCATGGACTAATTAGACTCAAAAGATTTGTCTCGCGATTTCCCCCCTAACTATACAATTATTTTTTAAATTTATATTTAATGCTCCATGCATGTGTCCAAAGATTCGATATAATGTTTTTTGGAAAAATTTTTTAGAACTAAACTAGGCCTAAGAGGAAGGTGATAAGTATCTA
3. Selection of Regions for Primer Design
Specific regions within the DRO1 sequence were identified and highlighted for primer development. These selected regions were used to design primer pairs with the objective of obtaining overlapping amplicons covering the target sequence.
The highlighted sequences represent the regions selected as potential primer-binding sites.
4. Overlapping Primer Design
A set of overlapping primers will be designed to amplify the complete target sequence rather than amplifying only the sequences located at the extreme 5′ and 3′ ends.
The primer-binding regions were identified using green-gradient highlighting in the sequence. Overlapping primer pairs will allow the complete target region to be amplified through multiple overlapping PCR products.
5. Basic Criteria for Primer Selection
The following criteria were considered for selecting the best primer combinations:
GC content: The GC content should preferably be within the range of 40–60%.
Melting temperature (Tm): The melting temperature should be compatible with the selected annealing temperature.
Any_th: The value should be 0.00.
3′ Th: The value should be 0.00.
These parameters will be considered collectively when selecting the most suitable primer combinations for amplification of the target DRO1 sequence.
