How to make more DNA

After isolating a single cell, the next step is some kind of DNA analysis.  That means you need DNA.  Yes a single cell has DNA but the amount of DNA in a single cell isn't enough for most types of analysis.  So, you need to make more of that cell's DNA. 

The way to make more DNA from a single cell is to perform whole genome amplification.  Each time a cell divides, it performs essentially whole genome amplification to ensure that the newly made cell has its own genome.  This whole genome amplification takes place within the cell using its own suite of enzymes.  We have to perform a whole genome amplification in a tube where the entire contents of the cell have been spilled out. 

Currently, the best way to do whole genome amplification in a tube is by using the enzyme Phi29 DNA polymerase.  A DNA polymerase is an enzyme that makes more DNA.  Phi29 is a specific DNA polymerase that replicates by a process called strand displacement.   

http://en.wikipedia.org/wiki/File:MDA_reaction_1.JPG

 

In strand displacement, random primers tell the polymerase where to begin making DNA.  The polymerase amplifies DNA for about 7 to 10 kb or until reaching a previously amplified region.  When the polymerase hits this previously amplified region, it actually displaces it and continues to make more DNA.  Meanwhile, new primers and new polymerases can begin amplifying DNA using either the original DNA or the newly replicated DNA.  The result is the branching network of DNA molecules shown in the picture above. 

The advantage of strand displacement is that it does a better job of replicating the entire genome uniformly and to high copy number than other amplification methods. 

At the very end of the entire process, all of the branching molecules are cut so that only linear molecules remain.  These molecules are then ready for DNA analysis!




FOOD!

Kinmedai (golden eye snapper)

Squid ink noodles

Modanyaki (savory pancake with noodles)

Sashimi

Mont blanc (chestnut cream dessert)

There's more than one way to dissect out a microbe.

Micromanipulation is just one way to isolate a single microbe.
This past week, I learned about two other methods used for single cell genomics.


The first was laser microdissection.

Laser microdissection is exactly what it sounds like.  You use a laser to cut out the cell you want.

http://www.leica-microsystems.com/products/light-microscopes/life-science-research/laser-microdissection/details/product/leica-lmd7000/

The part you cut out literally falls down onto a collection plate.  This might sound hard but it's not.  The laser is extremely precise and very easy to control using its computer software.  What the microscope sees is shown on the computer screen.

Find your cell of interest.

Then you just use your mouse to draw around what you want to cut out, tell the laser to go, and it's done!

Draw a circle around it.

Tell the laser to cut it out.

The collection plate with the part cut out.

While it took me about 15 minutes to micromanipulate a single cell, it took me only a few minutes with the laser dissector. 


The second technique for single cell genomics I learned about was cell sorting.

The cell sorter is a device that forces cells to move one at a time past a laser beam.  The device then records how long the cell took move past the laser and how the light from laser was refracted by the cell.  These pieces of information can be related back to the size, shape, and quality of the cell.  Most of the time, the cells being sorted have been made to fluoresce.  So the device will also measure the fluorescence coming off of the cells themselves.  Together, a fingerprint for each cell type is created.  So from a mixed population of cells, the device can type each cell and sort them apart.

Before sorting

After sorting

Cell sorting is an extremely powerful yet difficult method.  My lab was able to sort 200,000 cells in a matter of minutes.  However, it took us 8 hours to set up the device to read and sort the cells the way we wanted.  Additionally, we must do further checks to confirm the cells were correctly sorted.





The ocean, mountains, and a castle!

Pacific Ocean on the left, Mt. Fuji straight ahead

Odawara Castle

Odawara Castle

Odawara Castle

How to get a single cell

The key to my summer research is isolating a single bacterial cell.  There are a variety of ways to do this.  The primary way my lab does this is by micromanipulation -- that is, physically picking up a single bacterial cell and moving it away from everything else.  Micromanipulation is done using a manipulator attached to a microscope.  Here's one of the micromanipulators in lab:

If you look carefully just slightly above and to the left of the microscope stage, there's something that looks like a metal rod.

micromanipulator with capillary tube

That is the micromanipulator.  At the end of the rod is a very small glass tube called a capillary tube.  The tube is attached to a hollow rod that filled with first air and then further up oil.  The rod then continues as tubing that comes over to the left side of the microscope.  On that side, you can see a much bigger tube, a pump, with a knob on the end.

the pump

That pump allows you to change the pressure inside the capillary tube.  What this means is that if you turn the knob one direction, you can pull liquid into the capillary tube.  If you turn the knob the opposite way, liquid is expelled.  Basically, that thing is a very carefully controlled straw.

So the micromanipulator can suck up things.  But only very small things since the capillary tube is so small.

Next, how do you select what to suck up?  The manipulator can be moved around the stage of the microscope using a joystick.

the joystick

Yes, its just like any video game joystick.  With the joystick, you can move the micromanipulator in three dimensions. 

Let's put this all together.  On the microscope stage, you put a small puddle of liquid with the broken up termite gut.  Then you move the micromanipulator so that the capillary tube is positioned just next to the cell you like.  Next, you turn the knob on the pump and suck up your cell.  Finally, you move the micromanipulator to a clean puddle and drop your cell down.  Repeating this process a few times with the same cell washes it clean.

Your cell is then ready to have its DNA played with! 


The big and small of Tokyo!

Tokyo Tower

Sky Tree from Tokyo Tower

performer monkey

lake in my neighborhood

Imperial Palace grounds