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
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.