How to isolate DNA

How to isolate DNA

Today, I'm going to present a method of a very exciting experiment. It strongly ties in with what I have been discussing over the past week: DNA. Doing a biochemistry degree involves a lot of experimentation and this experiment is what I hope to be doing frequently during my education :)

The method of isolating DNA that I will present is the same across many parts of the world and is one of the most essential experiments in molecular biology.



Alright, gimme the main jist of it...

Okay, first we need to get an organism. I used an onion. Yes, it was a teary experiment. :')

We break up the tissue and use detergent to break down the cell surface membranes of... the cells and the nuclear membranes of the... nucleus. This is homogenisation.

The cell fragments are separated through filtration and we are left with DNA and soluble proteins.

To remove the protein, we use enzymes. Protease is a wise choice.

Finally, the DNA is precipitated using ethanol at around 250K (I personally kept it at 273K).

Apparatus

Liquidiser (mixer)
Knife and chopping board to cut onions
Water both that is ice cold
Thermometer
Funnel
Filter paper
500cm^3 Beaker
Glass rod
Stopwatch
Boiling tube and rack
25^3 measuring cylinder
Pipette
Sodium chloride solution (I used 4%M)
Low temperature ethanol (I used 95%M)

Detailed method please!

1. Slice onion with a knife into 5mm cubes and place in beaker with 100cm^3 detergent.

2. Stir and keep at 333K in a water bath for 15 minutes. The temperature breaks down the cell membranes of the onion and the detergent forms complexes surrounding the membrane phospholipids and proteins causing them to solidify out of the colution. Also, the Na+ sodium ions shield the negatively charged phosphate groups of DNA molecules causing them to coalesce.

(By this point, you will be crying. I shall give you the biochemical reason for this next time!)

3. Cool the mixture in iced water for 5 minutes whilst stirring. This slows down any breakdown of DNA if you kept it in high temperatures any longer.

4. Liquidise the mixture for ONLY 5 seconds - yeah it's that quick. This degrades the cell walls and membranes which allows more DNA to be released. The reason why liquidisation should be quick is because this stops the DNA strands from being completely and utterly broken up, which kinda destroys the experiment...

5. Filter it into a measuring cylinder and the filtrate should contain the DNA and soluble proteins.

6. Store this in a fridge for around 1 or 2 days

7. Add 1cm^3 enzyme to 20cm^3 of the onion tissue extract in a boiling tube and mix. The enzyme will hydrolyse the proteins associated with DNA.

8. Induce a layer of ice-cold ethanol on top of the mixture by pouring it slowly down the SIDE of the boiling tube.

9. Leave the tube for 3 minutes. The DNA is insoluble in ice-cold ethanol. Bubbles may form and the DNA will precipitate out.

10. Gently rotate the glass rod in the liquid at the interface of the alchol/detergent mix. The interface is a flash term for the point at which the two layers meet. Be careful not to stir too much as the mixing of layers would break up the fragile strands of DNA ~

11. A white web of DNA (which would look a lot like mucus) can be viewed. It can be drawn from the tube with a pipette and suspended again in a solution of sodium chloride or distilled water.


I hope this experiment helps people who may be getting ready to do their EMPAs or ISAs in the UK. This will help anyone who has a more practical approach to this very practical science of Biochemistry. Next week, inspired by my crying during when I carried out this experiment, I'm going to explain why we cry when we chop onions (and maybe a few old-wives tales too!).



Exocytosis

The Human Genome Project

The Human Genome Project

It's a famous project. You've probably heard of it. Wanna know more?

This legendary project began in 1990 and was planned to be a 15-year project. But technology sped up somewhere in between and we managed to finish it in 2003 - 2 years before the target date. There were a few objectives of the project:
      1. Locate and identify all the genes in human DNA - there's nearly 25000 of them!
      2. Find out the sequences of the 3 billion pairs of organic bases that make up the most
          important and influential part of DNA
      3. Store all the information in a single database that can be easily accessed internationally
          and be used for future references.

The picture below is the official logo for the Human Genome Project.


That's all great, but what's a genome?

Yeah, what's so special about them anyway? Well, a genome is all of an organism's hereditary information. It is found in the DNA of an organism such as a human or plant cell. In viruses, the genome can be found in its RNA (ribonucleic acid). Viruses are quite weird aren't they? They are living organisms right? Or are they? (yeah, I'll post a virus-specific blog soon!)

In my earlier, I told you about the pairs of bases that can exist in DNA. Well, altogether there are around 3.3 billion pairs of them that extend throughout an entire genome. Phew!



Above, you can see a nice picture. It is actually the initial result of the identification of the human genome. Where is this place? London baby! :)

Important benefits

This is a biochemistry blog (apparently) so I thought I should just outline the biochemical benefits of the Human Genome Project for interest's sake.

Many scientists have anticipated a new era in combatting disease. With the recent advancements in molecular medicine, we can now treat the fundamental causes of disease rather than simply relieving the symptoms which, to be honest, we've been doing since the Middle Ages.

If we can identify the variants in specific genes that increase the risk of cancers and genetic diseases, then we could potentially wipe them out in humans. If these variants can be cross-compared with other animals, it would help near-extinct organisms repopulate, increasing biodiversity.

Using gene therapy (the administration of DNA to treat disease), we could change genes that cause disease. We can go even further - replace the defective gene itself with a ''normal'' one, eliminating the risk of disease.



What have we learned?

So far, the project has shed some light on many issues that scientists have tried to confront. However, we can now safely confirm a few things:
      1. An average gene contains 3000 organic base pairs
      2. The human genome sequence is the same for 99.9% of the human population
      3. The human genome is mainly composed of the C and G bases
      4. The Y chromosome has the fewest genes compared to the other chromosomes - sorry guys!

The project was completed in 2003, but there is a worldwide post-sequencing project that is happening right now. The information we will gather in the next few years will provide us with knowledge that we can apply to areas of biology like human health, the environment and biodiversity.

So that's the basic gist of the Human Genome Project - now impress your mates. I've just realised that I've made three posts in two days when my target was one post a week. I guess I get the next two weeks off then!

Just kidding, watch this space - and don't forget to bombard me with feedback and comments!

Exocytosis

DNA and Nucleotides

DNA and Nucleotides

Okay, so I just came back from a biology lesson at school and we were discussing the structure of DNA. It isn't made up solely of proteins, carbohydrates or lipids. It's actually made up of nucleotides. At first, it may seem easy to remember but I was told that many people have strayed away from the beaten path regarding this.

What is DNA?

It's stands for deoxyribonucleic acid - can't imagine why they shortened that.
DNA is a chemical that determines genetically inherited characteristics in all living organisms. It is able to store vast amounts of information as a genetic code - hmm... what if we could use DNA to somehow store and access large amounts of information? It would be more compact and easier to transfer than our usual everyday data storage systems... Anyway, back to the topic at hand!

Frankly, DNA is ''sick'' - i.e. it's great. It's better than great: it is good (yeah, I reference Friends episodes from time-to-time, bear with me!). It is able to pass on characteristics between different organisms of adjacent generations - amazing.

Dude, I meant What is DNA?

Piccy time!


I'm sure you recognise it! I went to a lecture about DNA the other day: this helical structure is world famous - there are statues designed to look like this; there are coins that have this image symbolically engraved at the front: why? Because they look cool.

Biochemical components

One strand of DNA is made up of nucleotides.

One nucleotide (a mononucleotide) is made up of 3 components:
          1. Deoxyribose sugar (C5H10O4)
          2. Phosphate group (PO43-)
          3. Organic base



There are 2 base groups:
          1. Single-ring pyramidines: cytosine (C) and thymine (T)
          2. Double-ring purines: adenine (A) and guanine (G)

When I first heard of cytosine, I confused it with cysteine - an alpha amino acid. I had to look at the first rule: DNA is made up of nucleotides - not proteins, carbohydrates or lipids!

Forming a double helix

Look at the nice picture of the DNA. There are little coloured lines in between those thick twisting tubes. They are the organic bases I mentioned earlier. Basically, they are base pairs that join up together. One base group always bonds with a base in the other group.

E.g. a purine like adenine will only bond with a pyramidine like thymine.

Why should this happen? Basically, each rung of the DNA ladder must be the same length. It is always three rings in length. So a double ring will join with a single ring so that the total length is always 3 rings across. Pretty neat, huh? 

HOWEVER, adenine, a purine, will not bond with cytosine, a pyramidine. Why? I mean, cytosine is a pyramidine... confusion much?! :(

If you know a lot about enzymes then you know that substrates are complementary in shape to them. In the same way, antigens are complementary to antibodies. Again, in the same way:

          A is complementary to T and forms two hydrogen bonds
          G is complementary to C and forms three hydrogen bonds

In chemistry, hydrogen bonds are amazingly strong intermolecular forces.
In biology, when compared to disulfide bridges in an antibody (and cysteine and methionine proteins) and ionic bonds, hydrogen bonds are proper weak.

DNA is a really confusing topic - people have told me that the main reason is because they sometimes forget the basics or didn't understand DNA from the start. It's really hard but maybe reading this blog has helped you!

This post is mainly a response to my fellow students who told me that they were finding it difficult to comprehend so much in a single hour's lesson - so I hope this helps anyone trying to understand the fundamentals of... well... life!

I'll try to find some articles that concern DNA before my next post. Don't forget to give me feedback too :)

Exocytosis

Greetings!

Greetings! It's thebiochemist here.

This blog has been designed by myself, thebiochemist, in order to project, on a weekly basis, my thoughts on what I personally think is a very tasty subject: Biochemistry. It is a subject close to my heart...literally. As I endeavor to write my first EVER blog, I hope to learn something and maybe teach you (if you're unfortunate enough to lay your eyes upon this blog) something too!

I'm thinking about discussing current events that are circulating this topic which features at the very edges of biology. I'm a student so I hope to convey this in a non-formal way too!

For many years I have been wondering what makes life tick. What allows us to function as we do? Not only us: what allows all living organisms on this planet to carry out the process that they do?

I believe biochemistry can provide answers.

What is Biochemistry?

God knows.

Biochemistry is the study of chemical substances and process within the cells of living organisms. As you can probably tell by its name, it involves elements of both biology and chemistry so it's quite a demanding subject - which kinda makes it all the more exciting, right? :)

If you're a medicine, biological science or biophysics student, you're probably more focused on organisms, organ systems and individual organs as a whole. The amazing thing about biochemistry is that (it's better) you get to dig deeper - into a whole new world of molecules (see? this is where the nitty-gritty chemistry stuff spills in!).

What is a Blog?

I don't know actually... This is my first... I don't even know exactly what to include in it! Please send me some comments and feedback - I'm in a serious deficit and need of them.

Alrighty then, that's more than enough from me this week. I'll be sure to leave time to commit to these blogs every week. If I don't, I shall boldy face the biochemical consequences.

Exocytosis