Bio 3950 - Biotechnology                                                                                            April 2024  Dr Carr

Restriction Endonuclease Analysis

Purpose I: Design a set of overlapping PCR primers that will amplify the complete circular mtDNA genome of the Honey Bee, Apis mellifera mellifera.

Purpose II: Construct Restriction Maps of several subspecies of A. mellifera likely to be found in Newfoundland. Identify Restriction Endonucleases that could be used to identify species, based on PCR fragment data in Part I.

1) Retrieve Apis mellifera mellifera voucher 1410 GenBank accession KY926884

     Save as: Complete Record / FASTA sequence
     Retrieve mtDNA genomes from [two] other subspecies of A. m. mellifera by accession numbers, as instructed.
     Retrieve Cox1F - Cox2R amplicon

2) Open MEGA: Click on ALIGN icon; choose Edit / Build; select "Retrieve a sequence from a file";
                                                                                             select KY926884.fasta and the other *.fasta files.

The A. m. mellifera primers ApisCox-1F and ApisCox-2F span a region near the 5' end of the genome:  

                Three tRNA genes: last one tRNA-Trp runs         [1722 - 1793] =     71 + 1 =     72 bp
            Second ORF: "cytochrome c oxidase subunit 1"      [1794 - 3354] = 1560 + 1 = 1561 bp
                One tRNA gene: tRNA-Leu runs                          [3355 - 3424] =     69 + 1 =     70 bp
            Third ORF
"cytochrome c oxidase subunit 2"         [3618 - 4295] =   677 + 1 =   678 bp
                 Two tRNA genes: tRNA-Asp runs                        [4294 - 4362] =     68 + 1 =    69 bp

             Region spans [1722 - 4362] = 2640 +1 = 2,641 bp

Identify Restriction Sites with RestrictionMapper v.3. Construct a contig map for a subset of these as directed. [Alternatively: map a 4 Kb region, and assemble maps across Groups].

4) From the list of A. mellifera subspecies GenBank accession numbers in Table 1 of Carr (2023) assemble in MEGA a three-way alignment, for A. m. mellifera and [two] other subspecies as directed.
    1) Assemble by eye
    2) Assemble by CLUSTAL, with parameters TBD

    3) In the alignment, identify Restriction sites that would let you distinguish the subspecies in your set.




All text & Figure material © 2024 by Steven M. Carr