Diptera is an order of the class insecta that consists of 2 winged flies or so-called true flies.

Diptera is an order rich in species as it includes around 240,000 species. In this lab, we will catch

a Diptera and identify it. I recognized that the fly I caught is of the species Musca autumnalis

since the fly exhibited an orange and black patterned abdomen, black eyes, and hairlike

extensions. We will verify our identification by working with mitochondrial DNA. since it is

highly polymorphic, mutates rapidly, and since it is easy to isolate it. Therefore, it’s easy to

identify different species based on the differences in their mitochondrial DNA since it varies

between different species and is conserved between the same species. After its extraction, a

specific gene can be amplified and studied. Amplification can be done using PCR (polymerase

chain reaction) starting with a known primer and template (our extracted dna) and ending with

2n for each gene present where n is the number of cycles done. We perform about 30-45 cycles

where each cycle consists of denaturing the double stranded dna at high temperature, annealing

the primers on the single stranded dna, then allowing the taq polymerase to extend the other

strand at its optimal temperature. The amplified gene can then be cut using restriction enzymes

that cut at specific sites. Tru9 I is a restriction enzyme which cuts at TTAA sites between the

first and second T (T/TAA), as this sequence is highly repeated in the gene of interest. Based on

these cuts, we can identify different species of sequences mutated at these sites using (RFLP)

Restriction Fragment Length Polymorphisms method. The fragments then can be separated on an

agarose gel electrophoresis based on length by applying an electric current, the ones of smaller

size while migrate further than the larger sized ones. Sizes can be determined using a DNA size

ladder used as a standard. However, one limitation of this method is possibility of losing small

fragments.
For the purpose of identifying our fly, we will be extracting its mitochondrial DNA, amplifying

its mt-ND1 gene, cutting it using restriction enzyme Tru9 I, and running our fragments over gel-

electrophoresis, identify their sizes and try to compare our results to known mitochondrial

sequences of other flies whose mitochondria has been sequenced completely, as well as that we

can compare our fragments to that well know Drosophila melanogaster’s mt-ND1 gene.

However, a limitation is that not all flies have their mitochondrial DNA sequenced. In addition to

that RFLP method allows us to identify flies based on fragment sizes and not based on the actual

DNA sequence, thus not all mutations can be detected. Usually, such experiments aim at a better

understanding of different species of the same order, how they diverged, and to determine the

phylogenetics relationships between different species

Wael Osman
BIOL 223 Lab – Section B6
Ms. Rouba Hilal
October 29th 2020
Fly Mitochondrial DNA Extraction – Homework
I. Differences between the sequences of the horn fly (Musca autumnalis’s closest

relative) and Drosophila melanogaster

In the prelab report:

 The digestion by Tru9 I for the mtdND1 gene sequence of the closest relative to Musca

Autumnalis, namely horn fly (Haematobia irritans irritans), occurs at 10 restriction sites and

results in 11 restriction fragments.

The predicted fragment sizes were: 60bp, 5bp, 97bp ,32bp, 145bp, 21 bp, 15 bp, 158 bp, 15

bp, 120bp, and 22 bp.

 The digestion by Tru9 I for Drosophila melanogaster’s mtdND1 gene sequence occurs at 11

restriction sites and results in 12 restriction fragments.

1) 5’ATCATAACGAAATCGAGGTAAAGTTCCTCGAACTCAAATAAAAACAAAAGAA

ATAAAAGT 3’ (Size: 60 bp)

2) 5’ TAATTTTATATAAAT 3’ (Size: 15 bp)

3) 5’ TAATAAAT 3’ (Size: 8 bp)

4) 5’TAAACACATCACAAGGTAAAAAAATAACGCAAAATAATATTCTTATAAATAAA

ATTCTCGCATATTCAGCTATAAAAAT 3’ (Size: 79 bp)

5) 5’ TAAAGCCAAACCCCCTCTTCTATATTCTACAT 3’ (Size: 32 bp)

6) 5’TAAATCCTGAAACTAATTCTGATTCTCCTTCAGCAAAATCAAAAGGATTCCGAT

TAGTTTCAGCTAATGAAATAGATACTCAAACTAAAGCTATAGGAAATAAAATAA

T 3’ (Size: 109 bp)

7) 5’TAAAAATCATATATAAACTTGATAAAAAAAAAAATAAATTATATTATAACTTC

CAAT 3’ (Size: 57 bp)

8) 5’ TAAAAAAATAAAAGATAATAAAAT 3’ (Size: 24 bp)

9) 5’TAAAGCTAAACTAACTTCATAAGAAATAGTCTGAGCCACAGCTCGCAAACCTC

CTAATAAAGCATAATTAGAATTAGACGACCAACCAGCTACTATAACAGTATAAA

CCCCCAATCTAGTACAACATAAAAAAAATAAACCACCCAAAT 3’ (Size: 149 bp)

10) 5’TAAAAGAATATAATTTTACAAAAAAAGGCATACATATTCAAACAAATAATGAT

AAAAATAAAGAAAAAATTGGAGAAATATAATATCT 3’ (Size: 88 bp)

11) 5’ TAAATAATTAGATAATAAAGGATAAGTTTGTTCTTTTGTAAATAATT 3’ (Size:

47 bp)

12) 5’ TAATCGCATCACAAAAAGGTTG 3’ (Size: 22 bp)

 The 12 predicted fragment sizes were: 60 bp, 15 bp, 8 bp, 79 bp, 32 bp, 109 bp, 57 bp, 24bp,

149bp, 88 bp, 47bp, 22 bp.

II) Substitution mutations done on horn fly mtND1 gene sequence to obtain same

restriction fragment sizes as Drosophila melanogaster upon digestion with Tru9 I:

The specific nucleotides to be changed in the restriction sites are underlined and bolded.

We must also watch out for creating new restriction sites (with nucleotides before or after the

sites themselves) that we do not want when we substitute specific nucleotides.

Color legend:

- Yellow: Restriction sites that are identical in horn fly and Drosophila melanogaster and left

unchanged

- Blue: Old restriction sites only found in Horn Fly that will be changed to non-restriction sites

- Green: Restriction sites that were only found in Drosophila melanogaster and will be changed

to new restriction sites in Horn Fly

ATCATATCGAAATCGAGGTAAAGTTCCACGAACTCAAATAAATATAAAAGAAACA

AAAGTTAATTTAATATAAAATATAAAAGAAAATACATCTCTTCCTAAAAATATAAC

ACAAAATAATATTCTTATAAATAAAATACTTGCATATTCAGCTAAAAAAATTAAAGC

AAATCCTCCTCTTCTATATTCTACATTAAATCCAGATACTAATTCAGATTCCCCTTCA

GCAAAATCAAAAGGAGTTCGATTAGTTTCAGCTAAAGAAATTCTAAATCAAACTAA

TGCTATAGGAAATATAATAAATAAAAATCAAATAAATATTTGATATTTATAAAATAT

TAATATATTATAACCTCCAATTAAAAAAATAAAACTTAATAAAACCAAAGCTAATCT
AACTTCATAAGAAATAGTTTGAGCAACAGCTCGTAATCCACCTAATAATGCATAATT

TGAATTAGAAGATCATCCCGCAATTATAACAGTATAAACACCTAAACTTGTACAACA

TAAAAAAAATAATAACCCTAAATTAAAAGAAAATAATTTAATAAATATAGGTATAC

ATATTCAAACTAATAAAGACAAAAATAAAGAGAAAATAGGAGAAAAATAATAAGA

AATATAATTAGATAATAAAGGATAAGTTTGTTCCTTAGTAAATAATTTAATTGCATC

ACAAAAAGGTTG

4 old restriction sites only found in horn fly will be changed to non-restriction sites (4-point

mutations), and 5 restriction sites that were only found in Drosophila melanogaster will be

changed to new restriction sites in horn fly (9-point mutations in total).

→The 14 induced substitution mutations lead to the modified sequence below with 11 restriction

sites and 12 restriction fragments. The modified restriction sites are highlighted, and the specific

new modified nucleotides are bolded and underlined:

ATCATATCGAAATCGAGGTAAAGTTCCACGAACTCAAATAAATATAAAAGAAACA
AAAGTTAATCTAATATAAATTAAAAAATTAAATACATCTCTTCCTAAAAATATAACA
CAAAATAATATTCTTATAAATAAAATACTTGCATATTCAGCTAAAAAAATTAAAGCA
AATCCTCCTCTTCTATATTCTACATTAAATCCAGATACTAATTCAGATTCCCCTTCAG
CAAAATCAAAAGGAGTTCGATTAGTTTCAGCTAAAGAAATTCTAAATCAAACTAAT
GCTATAGGAAATATAATAATTAAAAATCAAATAAATATTTGATATTTATAAAATAAT
AATATATTATAACCTCCAATTAAAAAAATAAAACTTTATAAAATTAAAGCTAATCTA
ACTTCATAAGAAATAGTTTGAGCAACAGCTCGTAATCCACCTAATAATGCATAATTT
GAATTAGAAGATCATCCCGCAATTATAACAGTATAAACACCTAAACTTGTACAACAT
AAAAAAAATAATAACCCTAAATTAAAAGAAAATAATGTAATAAATATAGGTATACA
TATTCAAACTAATAAAGACAAAAATAAAGAGAAAATAGGAGAAAAATAATAAGTT
AAATAATTAGATAATAAAGGATAAGTTTGTTCCTTAGTAAATAATTTAATTGCATCA
CAAAAAGGTTG
Upon digestion of the modified sequence mtND1 gene sequence of horn fly by Tru9 I at 11

restriction sites (T*TAA), we obtain 12 restriction fragments, which are:

1. 5’ATCATATCGAAATCGAGGTAAAGTTCCACGAACTCAAATAAATATAAAAG

AAACAAAAGT 3’ (Size: 60 bp)

 2. 5’ TAATCTAATATAAAT 3’ (Size: 15 bp)

3. 5’ TAAAAAAT 3’ (Size: 8 bp)

4. 5’TAAATACATCTCTTCCTAAAAATATAACACAAAATAATATTCTTATAAATA

AAATACTTGCATATTCAGCTAAAAAAAT 3’ (Size: 79 bp)

5. 5’TAAAGCAAATCCTCCTCTTCTATATTCTACAT3’ (Size: 32 bp)

6. 5’TAAATCCAGATACTAATTCAGATTCCCCTTCAGCAAAATCAAAAGGAGTTC

GATTAGTTTCAGCTAAAGAAATTCTAAATCAAACTAATGCTATAGGAAATAT

AATAAT 3’ (Size: 109 bp)

7. 5’TAAAAATCAAATAAATATTTGATATTTATAAAATAATAATATATTATAACCT

CCAAT 3’ (Size: 57 bp)

8. 5’ TAAAAAAATAAAACTTTATAAAAT 3’ (Size: 24bp)

9. 5’TAAAGCTAATCTAACTTCATAAGAAATAGTTTGAGCAACAGCTCGTAATCC

ACCTAATAATGCATAATTTGAATTAGAAGATCATCCCGCAATTATAACAGTAT

AAACACCTAAACTTGTACAACATAAAAAAAATAATAACCCTAAAT 3’ (Size:

149 bp)

10. 5’TAAAAGAAAATAATGTAATAAATATAGGTATACATATTCAAACTAATAAA

GACAAAAATAAAGAGAAAATAGGAGAAAAATAATAAGT 3’ (Size: 88 bp)

11. 5’ TAAATAATTAGATAATAAAGGATAAGTTTGTTCCTTAGTAAATAATT 3’
(Size: 47 bp)

12. 5’ TAATTGCATCACAAAAAGGTTG 3’ (Size: 22 bp)

→ Thus, after inducing 14-point mutations in the mtdND1 gene sequence of horn fly which

changed 4 restriction sites unique to horn fly to non-restriction sites and created 5 new

restriction sites in horn fly that are originally only found in Drosophila melanogaster, we obtain
a modified sequence with 11 restriction sites. Upon digestion with Ttu9 I restriction enzyme, we

will obtain 12 restriction fragments of lengths: 60 bp, 15 bp, 8 bp, 79 bp, 32 bp, 109 bp, 57 bp,

24bp, 149bp, 88 bp, 47bp, 22 bp.

→ We will obtain the same number and length of restriction fragments upon digesting the

modified mtdND1 gene sequence of horn fly with Tru9 1 as the digestion of the mtdND1 gene

sequence of Drosophila melanogaster with Tru9 I.