Developing a Developmental Biology Lab – Roundabout Sex Determination

Welcome to my series documenting my trials and tribulations as I foray into academia.  I’ve recently been put in charge of creating and managing a series of labs for a college-level course in developmental biology.  The description of the course is below:

 

“This is a study of the principles of development and their application to animal and plant embryos, regeneration, metamorphosis, cancer and related processes. The laboratory includes observation and experimentation with living animal and plant material, plant tissue culture, and examination of prepared slides.”

 

I do have a colleague handling the corresponding lecture, but ultimately the responsibility for the labs falls on me.  Labs are three hours long and are held once a week for 13 weeks total. 

 

Designing this course has been fun, but it’s also a hell of a challenge.  The course hasn’t really been run before and most of the labs have to either be created from scratch or modified from protocols found within the Bio department or by searching the internet.  It's also hard to think of an appropriate lab that’s specific to developmental biology, but doesn't overlap with typical labs in a molecular biology, genetics, or anatomy course and also fits within three hours.  I haven't been able to find many resources online for creating this type of course so I thought I'd post my labs on SteemSTEM after I make them.

 

This week's lab continue's the last labs work on the human placenta.

PCR screening placenta tissue for the Y-chromosome

In the last lab, students analyzed my child's placenta.  We had a anatomy lesson and students learned to both fix tissue and extract DNA from that tissue.  Although I told the students that the placenta was my kid's, I never told them the sex of my child.  This week the student's task was to use a PCR screen to tell me whether I had a boy or a girl. 

The image I gave them of my kid was specifically cropped at the waist so they couldn't tell if it was a boy or a girl.

They're specifically screening for a gene called SRY, short for sex-determining region Y, which is needed for the initiation of male sex determination in humans (https://en.wikipedia.org/wiki/Testis-determining_factor).  This gene is only located on the Y chromosome so if the students see this gene in their screen, they can conclude that I had a boy.

Below is the protocol I handed out to them prior to the lab.

Protocol 

Overview

In the last placenta lab, we began isolating DNA from placenta tissue samples.  Today we will finish the DNA isolation and run a simple PCR screen to determine the sex of the baby. 

 

Although few labs would ever bother genotyping a fully developed placenta, this screen is surprisingly similar to the use of cell free fetal DNA (cffDNA) for early non-invasive sex determination¹.  cffDNA refers to fetal DNA present in the mother’s bloodstream released by the placenta.  It’s continuously present in small amounts throughout the majority of a pregnancy.  Scientists have been aware of this phenomenon for a long time, but only recently have modern advances in genomic sequencing and PCR techniques made it possible to analyze this type of DNA.

¹ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526182/

 

Reading

If you haven’t, read last week’s assigned paper on sample collection for placental research (https://www.ncbi.nlm.nih.gov/pubmed/24290528).

If you are not familiar with cffDNA, check out the Wikipedia page to gain a passing knowledge (https://en.wikipedia.org/wiki/Cell-free_fetal_DNA).  This field of study has been skyrocketing in recent years due to advances in DNA technology.

 

Diagram showing cffDNA entering the bloodstream (https://embryoplus.gr/en/cell-free-dna-nipt/)

Materials

Taq 2X Master Mix

SRY primers (5 uM each)

dH₂O

Thermocycler

PCR tubes (200 uL volume)

 

Preparing Template DNA:

Last week, we used SDS to isolate DNA from placental tissue.  This process yields plenty of DNA, but SDS can inhibit polymerases used in PCR reactions.  SDS can be purified from these samples, but the process takes time.  Depending on the time and reagents available, we will either perform a precipitation, column purification, or simple dilution to prepare this DNA sample for PCR analysis. 

In addition to using SDS-derived placenta DNA, we’re going to grind a frozen placenta with a mortar and pestle, heat to promote cell lysis, and centrifuge to pellet cell debris.  This will result in a dirtier sample with lower DNA yield, but should still work for our purposes.  PCRs require very little amounts of input DNA and Taq is a robust polymerase so low levels of cell debris are not expected to interfere with the PCR reaction.

Every PCR reaction requires a positive and negative control.  Our controls will be cheek cells from male and female students. 

 

1. Put 50-100 mg of ground placenta in a sterile Eppendorf tube.  Add 100 uL dH2O.

2. To obtain control DNA, gently scrape the inside of a student’s cheek with the tip of a sterile P200 pipette tip. Twirl the tip in 50 uL H2O in an Eppendorf tube.  Do this to get cells for both a male control and a female control.

3. Incubate all tubes at 95 ^oC for 5 minutes, vortex for at least 15 seconds, then centrifuge at max speed for a minute.

4. Transfer the supernatants to fresh Eppendorf tubes.  They should all contain DNA suitable for PCR purposes.

 

Preparing PCR reaction:

The PCR reaction uses 2 primers designed to amplify a 254 bp segment of the SRY gene.  This gene is only found on the human Y chromosome.  Incidentally, an intact SRY gene is needed for the testes to develop.

SRY_fwd:  CAT GAA CGC ATT CAT CGT GTG GTC

SRY_rev: CTG CGG GAA GCA AAC TGC AAT TCT T

 

Standard PCR reactions using taq will usually follow the temperature changes below. 

STEP	TEMP	TIME
Initial Denaturation	95°C	5 minutes
30 Cycles	Denaturation: 95°C Annealing: 45-68°C Elongation: 68°C	15 seconds 15 seconds 1 minute/kb
Final Extension	68°C	5 minutes
Hold	4-10°C

The major variables for any PCR are the annealing temperature and the elongation time. 

1.)    Go to https://tmcalculator.neb.com and calculate the appropriate annealing temperature. 

Annealing Temperature:  _____

 

2.)     Determine the elongation time based on the size of the fragment and the speed of the polymerase. 

Elongation Time:              ______

 

Next, we need to create the PCR reaction mix.  We need mix for 4 reactions (both placenta DNA preparations plus the male and female control DNA samples), but we will create enough mix for 5 reactions to avoid running out of master mix.  Fill in the table below for the recipe.

 

 

When you finish, double check these values with your instructor.  Once approved, you can take the Taq and primers from the front of the classroom and create the mix. 

Add 23 uL PCR mix to 4 PCR tubes.  In each tube, add 2 uL of a template DNA.  Once complete, cap the tubes and store them in the refrigerator until everyone has finished.  We will start the PCR reaction as a group.

 

Final Thoughts and Results

This lab was easy for students to follow.  No step was too demanding and they easily fit everything within the 3 hour timeframe.  However, success was never guaranteed with this lab because I used a hackneyed approach to DNA isolation.  I should have used a kit designed to isolate DNA from tissue, but I didn't have one available.  Instead I used detergents and heat to break open cells and hoped that was good enough to obtain DNA.  In fact, I had some students further purify DNA extracted using SDS with plasmid miniprep columns.  They're not designed for genomic DNA isolation, but they were the only columns I had in lab. 

This turned out to be a good idea.  Because most of the PCR reactions failed, yet one of the placenta DNA samples purified with the column succeeded (P2 on the image below)

The only visible band is from a placental DNA extract.  This band is at about the expected size (~250 bps).  Just the fact that we have a band at this size at all tells us that the primers and polymerase worked fine.  Since my child is a boy, the results align with what we expect so we can call this screen a success.  PCR screens of the other DNA samples probably failed because the method for extracting DNA either didn’t yield enough DNA or contained significant contaminants that interfered with the PCR reaction.  

Still a single success is pretty good considering we cobbled this experiment together from scratch.