1. Introduction to DNA

2. The Central Dogma of Biology
Go thru central dogma
Include gene in DNA
Appllies universally to all living organisms
Yet so malleable we have diverse life living in almost every available niche on our planet
In our workshop, we will focus on content and techniques that will take us through this central dogma
Today we’ll start with an introduction to the structure of DNA and how it replicates

3. Structure of DNA
This slide shows the chemical structure of DNA. It is fairly complicated but elegant and I am going to work you through it
DNA is built from a series of nucleotides which are strung together.
One nucleotide consists of a phosphate, sugar, and nitrogenous base
Point out in diagram as purple circle, yellow diamond, and colored squares
The nucleotides are paired left to right and then strung top to bottom to form what looks like a ladder
The phosphate and sugar groups are purely structural and make up the sides of the ladder and are called the sugar-phosphate backbone.
Let’s look at the phosphate group in the middle. It has a central P and four Os and is negatively charged.
Let’s look at the sugar. The sugar is made of five carbons which are numbered 1 – 5 as you can see in this diagram.
The sugar is called deoxyribose because it doesn’t have an oxygen at carbon 2
Let’s look at how the sugar and phosphate connect in this bottom left diagram: You can see that one of the oxygens in the Phosphate group binds to the 5 carbon below it and the other binds to the 3 carbon above it. This connection is called a phosphodiester bond.
Let’s look at how the phosphodiester bonds are formed in the two sides of the ladder. On one side we start with an unattached phosphate group, which then attaches to the carbon 3 etc. while the other one goes in the opposite direction so we say one sside of the ladder goes 3 to 5 prime while the other is antiparrallel and goes 5 to 3 prime.
The nitrogenous bases are in the middle and they carrry the information in genes which we will talk about tomorrow. There are four bases cytosine, guanine, adenine and thymine, abbreviated as CGTA. The connect in what are called base pairs tvia hydrogen bonds o form the rungs of the ladder with C always to G and A always to T
DNAs most stable form is called B and it twists clockwise so is called a double helix.

4. DNA Replication and DNA Polymerase
DNA has to be replicated when cells divide to make new cells.
Examples?
The entire genome must be uncoiled and replicated exactly. It is a very complicated process I am going to go over briefly because I want you to learn about the most important aspect of it: DNA polymerase
Replication starts at multiple sites along the DNA molecule called origins of replication
At the ORI, DNA gyrase removes the supercoils and DNA helicase untwists the double helix and separates the left and right sides of the ladder
This creates a Y-shaped region of DNA called the replcation fork
Then, DNA polymerase attaches with several other factors and synthesizes a new matching strand.
\DNA polymerase always goes 5 to 3 prime – show in diagram so on one strand it can move straight along and this is called the leading strand
On the other it does several segments of 5 to 3 prime on the lagging strand and those segments are called Okazaki fragments which are connected together by DNA ligase

5. Polymerase Chain Reaction - PCR
PCR amplifies DNA
Makes lots and lots of copies of a few copies of DNA
Can copy different lengths of DNA, doesn’t have to copy the whole length of a DNA molecule
One gene
Several genes
Lots of genes
Artificial process which imitates natural DNA replication

6. How PCR Works Reagents Needed DNA sample which you want to amplify
DNA polymerase
Taq DNA polymerase – Works at high temps (explained in a minute)
Nucleotides
Called dNTPs
Pair of primers
One primer binds to the 5’ end of one of the DNA strands
The other primer binds to the 3’ end of the anti-parallel DNA strand
Delineate the region of DNA you want amplified
Water
Buffer

7. How PCR Works Protocol Put all reagents into a PCR tube
Break the DNA ladder down the middle to create two strands, a 5’ to 3’ strand and a 3’ to 5’ strand
Melting or heat denaturation
Bind each primer to its appropriate strand
5’ primer to the 5’ to 3’ strand
3’ primer to the 3’ to 5’ strand
Annealing
Copy each strand
DNA polymerase
Extending

8. How PCR Works Temperature Protocol Initial Melt: 94ºC for 2 minutes
Melt: 94ºC for 30 seconds
Anneal: 55ºC for 30 seconds
Extend: 72ºC for 1 minute
Final Extension: 72ºC for 6 minutes
Hold: 4ºC
cycles

9. How PCR Works

10. PCR Movie

11. Gel Electrophoresis of DNA
Gel electrophoresis detects the presence of DNA in a sample
Gel electrophoresis detects the number of nucleotides in a fragment of DNA
e.g., the number of nucleotides in a DNA region which was amplified by PCR
Is a rough estimate, is not exact, need more sophisticated sequencing techniques to get an exact number of nucleotides
Can be used to tentatively identify a gene because we know the number of nucleotides in many genes

12. How Gel Electrophoresis of DNA Works
A sample which contains fragments of DNA is forced by an electrical current through a firm gel which is really a sieve with small holes of a fixed size
Phosphate group in DNA is negatively charged so it is moved towards a positive electrode by the current
Longer fragments have more nucleotides
So have a larger molecular weight
So are bigger in size
So aren’t able to pass through the small holes in the gel and get hung up at the beginning of the gel
Shorter fragments are able to pass through and move farther along the gel
Fragments of intermediate length travel to about the middle of the gel
DNA fragments are then visualized in the gel with a special dye
The number of nucleotides are then estimated by comparing it to a known sample of DNA fragments which is run through the gel at the same time

13. How Gel Electrophoresis of DNA Works
Reagents Needed
Sample of DNA fragments
Known sample of DNA fragments
DNA ladder
Gel
Agarose
Dye to visualize the movement of the sample as it is traveling through the gel
Loading dye – Blue juice
So know when to stop so sample doesn’t just run out of the gel
Dye to visualize DNA after it has traveled to its final spot in the gel
Syber® Safe
Buffer

14. How Gel Electrophoresis of DNA Works
Equipment Needed
Box to hold the gel
Comb to create small wells in the agarose gel to put the DNA sample into at the beginning of the gel
Positive and negative electrodes to create the electrical current
Power supply
Gel photo imaging system

15. How Gel Electrophoresis of DNA Works