TITLE: Marine mammal sex determination using epidermal tissue recovered from suction-cup tags
AUTHORS: Sadie Novak, Jacob MJ Linsky, Kaitlyn J Knapp, Susan Parks, David Wiley, Dana Cusano

[DESCRIPTION]
For many marine mammal species, minimal sexual dimorphism means that sex cannot be reliably identified through observation. Instead, genetic analysis of sampled skin and blubber are required to determine an individual's sex. Studies using suction cup tags on free-ranging whales may retrieve exfoliated epidermal tissue from the underside of the tag’s suction cups, offering a valuable source of genetic information. This study provides a protocol for tissue collection and DNA extraction from recovered tag tissue and describe the accuracy of PCR-based sex determination using these methods. 

[STEPS]
SECTION: Field Sampling
1. Retrieve the tag from the water. While retrieving the tag, ensure that nothing touches the suction cups with the exception of the net used for retrieval and wear gloves to prevent contamination.
SECTION: Field Sampling
2. Using fresh nitrile gloves, wipe the inside of the suction cups with a sterile swab, avoiding the outside of the suction cup and any other part of the tag. This reduces the chances of obtaining a sample that is contaminated with the genetic material of another animal that may have come into contact with the tagged animal. Note that suctions cups may not have visible skin. 
SECTION: Field Sampling
4. Place the swab tip first into a plastic vial, trimming the handle as needed in order to close or screw on the lid.
SECTION: Field Sampling
5. Clearly label the vial with the date and the identifying information that is associated with that specific deployment. Make sure that there is enough information on the label to ensure that all samples are easily and accurately associated with the respective deployment data. This includes, at a minimum, the ID associated with the tag deployment. For example 'bb22_115a', indicating the two letter species ID (bb), julian day (115), and the order of deployment (a, the first tag of the day).
SECTION: Field Sampling
7. Repeat steps 3 through 6 for the remaining pair of suction cups.
SECTION: Field Sampling
6. Move the vial immediately into a -80 °C freezer if possible. If no -80 °C is available on board the vessel, store on dry ice until the sample can be moved into an appropriate freezer.

SECTION: Field Sampling
5.1. It is also important to note this information on a datasheet designed to store this data along with other metadata that will be needed later. Of particular importance are:
Date the sample was collected
Time the sample was collected
Location (latitude and longitude) the sample was collected
Group size/proximity to other animals, as these factors may be a source of contamination
SECTION: DNA Extraction
15. Add Buffer AW2 to each sample as directed by the manufacturer's protocol and centrifuge samples at 17000 x g for 3 min to completely dry the column membrane. Discard the resulting flowthrough and collection tube. Place spin column in a clean 2 mL collection tube (included in kit). 
 

SECTION: PCR Amplification
19. Prepare the following reaction mixture for each sample of interest in 0.2 mLPCR-grade tubes:
 
  DNA Template 2 µLForward SRY Primer (20µM).5 µLReverse SRY Primer (20µM).5 µLForward ZFX/ZFY Primer (20µM).5 µLReverse ZFX/ZFY Primer (20µM).5 µLMyTaq HS Red Mix, 2x12.5 µLWater (dH2O)8.5 µL

 

SECTION: PCR Amplification
19.1. Amplify DNA fragments of interest with 30 cycles of PCR at the following conditions:
  
    StepTemperatureTimeCyclesInitial Denaturation95 ℃3 min1Denaturation95 ℃15 sec35 Annealing55 ℃15 secExtension72 ℃20 sec

SECTION: Analysis of PCR product by gel electrophoresis
21. Cast a 2 % volume agarose gel dyed with SYBR Safe DNA gel stain.
SECTION: DNA Extraction
8. 
 
To begin DNA extraction, remove swab tip from plastic stick. This can be done by tracing a clean razor blade along the connection point from the swab tip to the swab stick. Transfer the swab tip to 
a clean 1.5-mL microcentrifuge tube for each sample.

 

SECTION: Analysis of PCR product by gel electrophoresis
20. The PCR product will be assessed by gel electrophoresis using the known length of the SRY (210-260 bpd) and ZFX/ZFY (442/445). This protocol is written for use with an EasyCast B1 Mini Gel Electrophoresis system (ThermoFisher).
SECTION: Field Sampling
3. Swab two suction cups with one swab, then the other two suction cups with a new sterile swab. Retrieving two samples from the same tag allows for more testing as well as sample security in cases of contamination.
SECTION: Analysis of PCR product by gel electrophoresis
22. Re-position gel tray with gel in the buffer chamber of the gel electrophoresis apparatus to the running position (wells should be placed in the chamber so that they are positioned at the negative electrode). Fill the buffer chamber with 1X TAE buffer so that the gel is completely submerged. Remove comb. 
SECTION: Analysis of PCR product by gel electrophoresis
24. Load 2.5 µL of 100 bp DNA molecular weight marker (New England Biolabs, cat. no. NEB#B7025) prepared to contain 1X bromophenol blue running dye into the first well (bromophenol blue runs at ~500 bp). Load 10 µL of the sample(s) in the following wells. 
SECTION: Analysis of PCR product by gel electrophoresis
25. Place the lid on the electrophoresis apparatus and ensure that cables are connected to the power supply. Run the gel at 80-100 V under dark conditions. Monitor gel run until the bromophenol blue running dye line has migrated about 1 cm from the midpoint of the gel (~45 min -60 min ).  
SECTION: Analysis of PCR product by gel electrophoresis
26. Once the gel run is complete, resolved DNA fragments may be visualized with a UV transilluminator.
 

SECTION: Product analysis
27. The expected molecular weight of the PCR product associated with the SRY gene is 210-260 bp and the expected molecular weight of the PCR product associated with the ZFX/ZFY gene 442-445 bp.

The presence of the SRY gene suggests that the DNA sample came from a male individual. Females will not possess this gene, and thus this DNA fragment will not be amplified in DNA extracted from a female. However, absence of this gene may be caused by issues in the experimental workflow, such as an insufficient amount of template DNA added, which would result in a false result. To validate the PCR reaction and minimize the chance a false sexing, DNA is also screened simultaneously for a gene that is present in both male and female individuals. The ZFX/ZFY gene serves as a positive control in this protocol. 

An expected profile for DNA extracted from a male individual would show the presence of DNA fragments from both the SRY gene (210-260 bp) and the ZFX/ZFY genes (442-445 bp). An expected profile for DNA extracted from a female individual would show presence of only the DNA fragement from the ZFX/ZFY gene (442-445 bp). A sample that showed presence of only the DNA fragment from the SRY gene would suggest non-specific priming and would be considered an inconclusive result.     


 

SECTION: PCR Amplification
18. 
 
PCR amplification was performed on extracted DNA samples using primers specific to the SRY (sex-determining region Y) and ZFX/ZFY (sex chromosomal zing-finger) genes. Oligonucleotide primers for both of these genes have been validated in other species. Primers were ordered from IDT DNA. 

Forward SRY: 5’ CCC ATG AAC GCA TTC ATT GTG TGG 3’
Reverse SRY: 5’ ATT TTA GCC TTC CGA CGA GGT CGA TA 3’

Forward ZFX: 5’ ATA ATC ACA TGG AGA GCC ACA AGC T 3’ 
Reverse ZFX: 5’ GCA CTT CTT TGG TAT CTG AGA AAG T 3’
SECTION: DNA Extraction
9. Add the amount of Buffer ATL directly to each tube containing swab as directed by the manufacturer's protocol. Pipette solution up and down and over entirety of swab thoroughly, at least 20 times. 
SECTION: DNA Extraction
10. Add the amount of Proteinase K to each tube containing the swab and previous buffer solution as directed by the manufacturer's protocol. Pipette the solution up and down over the swab to mix. Vortex each sample for 15 s on high to ensure thorough mixing.
SECTION: DNA Extraction
11. Incubate samples at 56 °C  in a hot water bath for 180 min.
SECTION: DNA Extraction
12. Add Buffer AL to each sample as directed by the manufacturer's protocol and mix thoroughly by quickly vortexing for 15 s. Immediately add 200 µL of 96 % (v/v) EtOH and mix again by vortex for 15 s.
SECTION: DNA Extraction
13. Pipette solution into the DNeasy Mini spin column and place column in a 2 mL  collected tube (included in kit). Incubate samples at Room temperature for 5 min before centrifuging samples at 6000 x g for 5 min.  Discard flowthrough and collection tube. Place spin column in a clean 2 mL collection tube (included in kit). 
 

SECTION: DNA Extraction
14. Add Buffer AW1 to each sample as directed by the manufacturer's protocol and centrifuge samples at 6000 x g for 5 min.  Discard the resulting flowthrough and collection tube. Place the spin column in a clean 2 mL collection tube (included in kit). 
SECTION: DNA Extraction
16. Place the dried spin column into a clean 1.5 mL eppendorf tube and apply  100 µL Buffer AE onto the center of the column membrane. Be sure not to touch the membrane with the pipette tip. Allow the buffer to incubate on the membrane for 5 min at room temperature. Elute DNA by centrifuging the column at 6000 x gfor 1 min. Elute DNA from the column again by applying 100 µL Buffer AE onto the center of the column membrane and centrifuging at 6000 x g for 1 min.

 

SECTION: DNA Extraction
17. Dry DNA samples in a speed vac overnight at room temperature. Re-solubilize DNA in 20 µL of ultrapure water. Measure sample concentration (ng/uL) and purity (260/280 value) using a Nanodrop. 
SECTION: Analysis of PCR product by gel electrophoresis
23. Dilute running dye in PCR product mixture to a final concentration of 1X xylene cyanol (runs at ~4000 kbp) by adding 4 µL  of a 6X xylene cyanol solution to each sample product.
SECTION: Analysis of PCR product by gel electrophoresis
21.1. Prepare mold by placing gel tray in the buffer chamber of the electrophoresis apparatus so that the gaskets of the tray form a seal with the walls of the chamber. Use a level to adjust the tray position so that the gel is cast evenly.
SECTION: Analysis of PCR product by gel electrophoresis
21.2. Prepare the 2% agarose solution by combining the appropriate mass of agarose with the appropriate volume of 1X tris-acetate EDTA (TAE) buffer (i.e. for a 75 mL agarose gel, combine 1.875 g agarose in 75 mL 1X TAE buffer).
SECTION: Analysis of PCR product by gel electrophoresis
21.3. Melt agarose by microwaving solution in 15 s- 30 s time increments, swirling flask between heatings.
SECTION: Analysis of PCR product by gel electrophoresis
21.4. Allow solution to cool at room temperature for 5 min before adding appropriate volume of SYBR Safe dye to the solution to dilute to 1X SYBR Safe DNA gel stain (i.e. for a 75 mL agarose gel, add 7.5 µL 10,000X SYBR Safe DNA gel stain to solution). 
 

 

SECTION: Analysis of PCR product by gel electrophoresis
21.5. Once agarose solution is stained, cast immediately in appropriate gel mold. Place a comb in the mold to create a row of wells. Allow gel to polymerize at Room temperature for 20 min under dark conditions to protect the DNA stain.