Introduction to DNA

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

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.

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

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

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

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

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 30-35 cycles

How PCR Works

PCR Movie

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

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

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

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

How Gel Electrophoresis of DNA Works