The Summer of Sequences

Summer 2017

 

Lipsett Lab: The Summer of Sequence

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Haoju

Studying gene mutations in large mammals is important because such mutations can be correlated with human genetic diseases. In this case, Dr.Lipsett’s lab is investigating a gene sequence of Fibroblast Growth Factor 5 (FGF5) gene in Scottish Highland Cattle.

Why Scottish Highland Cattle?

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Figure 1: Scottish Highland Cattle

What makes Scottish Highland Cattle special is its extremely long hair compared to other cattle. The abnormal growth of long hair might be the mutation of FGF5 gene which controls the hair growth. When a mutation happens in the gene, the signaling factor of hair grow cycle lost its function, causing hairs to keep growing without degenerating. Since scientists have proven the mutation of FGF5 gene in donkeys, dogs, and mice, finding a mutation of the FGF5 gene in cattle would be another step of progress. In addition, studying of the implication of genes in cattle helps us to understand abnormal cell growth in humans. The abnormal growth is correlated with abnormal cancer cell growth. By studying the mutation of genes that cause abnormal hair growth, therefore, unregulated growth factors, the abnormal growth of cancer cells can be further understood.

Techniques

For the past six weeks, experiments have been done on DNA samples donated by farmers who own Scottish Highland Cattle. Farmers signed the Vertebrate Animal Use Request form approved by IACUC to participating in the experiments and mail the hair back. Control samples come from cattle with short coats. By collecting the root of hair from samples and using a QIAgene kit, DNA samples were extracted and used for further experiments. As a Chemistry major student, I have learned two important techniques that I have never encountered in my general chemistry lab and organic chemistry lab; these two techniques are biology based.

Agarose Gel Electrophoresis (Running a gel)

Gel electrophoresis is an important lab procedure as it helps scientists to visualize the length of DNA sequences, the purity of PCR product when it comes to deciding further PCR purification.

The gel we used in the Dr.Lipsett’s lab is completely homemade by Celine and Haoju. Ingredients are listed below:

  1. 2 grams of Agarose
  2. 250 ml Erlenmeyer flask
  3. 100ml of 10 M TBE (Tris/Borate/EDTA)
  4. 10 microliter of Gel Red3

Figure 2: Preparation on Gel Electrophoresis.

The procedure is as easy as making a Jell-O. Mixing Agarose with 100 ml of 1X TBE and microwave the mixture for 2 minutes (Making sure it does not boil). Then, add 10 microliters of Gel Red and pour the solution into a Gel box.

When filling the well combs, we used ThermoScientific DNA ladders to help visualize the side of DNA sequence. DNA ladder is put on each side of the well combs. The small blue box in the picture below is filled with DNA ladder. The PCR products are green.

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Figure 3: Loading a gel with PCR products.

When everything is ready, the gel box is connected to a voltage source. The DNA samples in the well combs move towards the anode and in 20 to 25 minutes, the bands will move to an appropriate length and the UV image of the gel can be taken.

 Nanodrop

Nanodrop is a UV/vis spectrophotometer follows Beer’s Law:

A=εlc

A=absorbance, ε=molar absorptivity coefficient, l=path length, c=concentration.

In this case, path length equals to 1mm. Therefore, much smaller movement can be seen.

Another important technique that I have learned this summer is using Nanodrop 2000c to determine the concentration and quality of DNA samples. Thermo Scientific Nanodrop 2000c in Dr. Powell was used. By applying 1 microliters of DNA samples on the pedestal using a pipett and closing the arm, the instrument automatically determined the concentration of DNA samples, shown as nanogram per microliter, 260 and 280 ratios and a graph with curves on the screen. The 260/280 ratio is used to determine the quality, or in other words, purity of DNA samples. A number close to 1.8 is considered as a pure DNA sample. Knowing the concentration of the DNA sample helps calculate the proper amount of DNA samples mixed with water in order to send out the plate for further DNA sequencing.

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Figure 4: Haoju nanodropping purified PCR product

 

 

Celine

I am the fourth Gettysburg College student to be working on the Equine Deafness project in the Lipsett Lab. We’re looking for mutations in certain genes in order to correlate congenital deafness and loss of pigmentation in a family of Spanish Mustang Horses.

You can usually find Orange and I sitting in the fishbowl on the main floor of the Science Center, usually updating our lab notebooks, inputting data into excel spreadsheets or most importantly, searching for mutations in our DNA sequences.

Our days usually rotate in a pattern of preparing the samples to sent and then examining the nucleotides and tracking mutations. We begin all of our experiments by isolating DNA, using an extraction kit, from horse hair donated to the project by various Spanish Mustang owners around the country. We have both control and experimental horses in our database of DNA samples. This means we have both deaf with lack of pigmentation horses and hearing with lack of pigmentation horses. Our horses vary in lack of pigmentation.

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Figure 5: A Spanish Mustang and its foal.

Next we design primers for our polymerase chain reactions (PCR). Designing primers is very important because it enables us to replicate a specific DNA fragment millions of times in a thermocycler. After running a PCR reaction the samples are run on a gel. The samples that ran correctly on the gel are then purified and nanodropped in order to send for sequencing. This summer I’ve purified upwards of 200 sequences.

 

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Figure 6: Purifying PCR products in order to send samples for sequencing.

Once the sequences come back from being sequenced, I search through the various exons to find mutations in certain horse genomes. If a mutation is found, I mark it with a designated letter.

G/C –> s

A/T –> w

C/T –> y

A/G –> r

G/T –> k

C/A –> m

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Figure 7: A screenshot of the alignment tool, CodonAligner which enables us to track mutations in certain genomes.

We hope to use this research in the future to find correlations between loss of pigmentation and deafness in not only horses but someday humans. The loss of pigmentation and deafness correlation in humans is classified as Waardenburg Syndyrome, which is a rare genetic disorder.

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Figure 8: Celine and Haoju with Capser, Dr. Lipsett’s dog.

 

 

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