DNA Identification

How do you find out if a sample is contaminated or not?
Find out whose DNA you have.

Every individual has its own genome, but sequencing an entire genome just to ask whose DNA you have is not practical (at least not in 2011).  We can instead just sequence a small region of the genome that is a good representation of the identity of the individual.  The region we choose to sequence must be:
1) present in all individuals
2) different in all individuals

For bacteria, the gene encoding the 16S ribosomal RNA (rRNA) is the most commonly used sequence for bacterial identification.  The 16S rRNA gene has both sequence found in almost all bacteria as well as sequence found only in a single type of bacteria.  These regions are called highly conserved or hypervariable.

For sequence identification, we begin sequencing from the highly conserved region, using DNA primers that match the highly conserved region, and sequence into the hypervariable region.

When we get the 16S ribosomal RNA sequence results back, it is just a DNA sequence -- not human readable.  We can, however, match this sequence to known sequences in a DNA database.  The National Institute of Health maintains such a database (http://www.ncbi.nlm.nih.gov/guide/) and the program that does the sequence matching is called BLAST.

Here's an example of a sequence match:

BLAST results

This 442 base pair sequence matches perfectly to Pseudomonas trivialis. 





Hakone, an area formed by an ancient volcano, now a popular hotspring area!

cable car overlooking Lake Ashi

ancient volcano, Mt. Hakone

sulfurous fumes from Mt. Hakone

sulfur mine at Mt. Hakone

eggs being cooked in hot, sulfurous water

kuro tamago (black egg) after cooking

DNA is DNA

Whole genome amplification is a very powerful technique.  With a really major caveat. 

It's blind.

It will make tons of copies of any DNA available.  Your microbe's DNA, your DNA, your dust mites' DNA, your cat's dinner's DNA.  If even a trace of DNA ends up in your reaction, it gets amplified right along with your sample.  This is called contamination.

The more limiting your sample, the bigger a problem contamination.  So if you're sequencing DNA from ancient humans, you probably only have traces amounts of ancient DNA around and lots of contaminating present day human DNA.

What's the solution to dealing with contamination?  Be really, really, clean.  In fact, use a clean room.

A clean room is a room that is sealed off and is fed filtered air.  Clean rooms are classified by how many of how small particles are let into the room.  The clean room in my lab is class 1000, which means that a maximum of 1000 particles per cubic foot of size greater than 0.5 micrometers are allowed in. 

All of this seems reasonable until you consider that a person needs to be able to enter and work in the clean room.  Obviously, a person is constantly shedding hair, skin, and DNA.

To solve this problem, we suit up and then shower down!

All ready for the clean room

Here's the get-up we wear to enter the clean room.  The suit itself is stored in a UV cabinet.  Why UV?  Because UV kills DNA.  Baking everything in UV is an easy way to eliminate contaminating DNA.

After getting all dressed up, you enter the first door to the clean room.  Inside is a small area that showers you with air.  The point is blow away contamination.  Finally, you go through a second door and are finally inside the clean room!





A trip to the old part of Tokyo, Asakusa!

Kaminarimon, Asakusa

Sensō-ji, Asakusa

Sensō-ji, Asakusa

Asakusa

To Japan's Past and Science's Future

Last week I attended an evolutionary biology conference in Kyoto.  The conference featured a wide variety of talks including:

-why our genes are encoded by DNA and not RNA
-how genes die
-the origin of mitochondria
-the stinkbug microbiome
-photosynthetic slugs
-the origin of eukaryotes
-the effect of diet and host on the primate microbiome
-horse domestication
-ancient bacterial DNA from teeth
-the effect of host living temperature on the genome
-how centromeres are lost
-the environment of the ancestor of all present life


Before and after the conference, I traveled around two of Japan's previous capitals, Nara and Kyoto.  Nara was the capital of Japan from 710 to 784.  A deer is said to have led a god to find and establish Nara as the capital and so deer are protected animals found all over Nara Park.

Nara Park

Along Nara Park, the largest wooden building in the world with the world's largest bronze statue of Buddha are found.

Todai-ji

Daibutsu

Kyoto was the capital from 1180-1868.  Kyoto is a city rich in culture, art, and food.  Many of its temples and shrines are world relics and frequently appear in film.

Mt. Daimonji

This character, dai, is one of five sites set afire on August 15th to honor the spirit world.

Fushimi Inari Shrine

Fushimi Inari Shrine is the central shrine to the god Inari in Japan.  This shrine grounds are covered in some thousand red torii gates.  I spent 2 hours walking through torii gates!

Kiyomizu-dera

Kiyomizu-dera is the temple of "clear water" situated above the trees, overlooking the city.  It is said that if one jumps and survives the 13 meter fall from Kiyomizu-dera, their wish will be granted.

I didn't jump... but I did have my wish granted of experiencing Kyoto kaiseki.

Kikunoi kaiseki first course

Kaiseki is traditional Japanese court cuisine similar to the western tasting menu.  The meal runs about 8 to 14 courses with each course featuring seasonal and exquisite foods presented in elegance and modesty.  A kaiseki experience leaves you filling reborn and heavenly.