Introduction to DNA, PCR and Gel Electrophoresis

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

Key enzymes involved in DNA Replication DNA Polymerase - uses each strand of separated DNA as a template to synthesize new strands of DNA with the precise, complementary order of nucleotides. DNA Ligase – an enzyme, that facilitates the joining of DNA strands together  Primase - DNA Polymerase cannot initiate the synthesis of DNA, primase is the enzyme that can start an RNA chain from scratch Helicase - untwists the two parallel DNA strands Topoisomerase - relieves the stress of this twisting Single strand binding protein - binds to and stabilizes the unpaired DNA strands

DNA Replication vs. PCR PCR is a laboratory version of DNA Replication The laboratory version is commonly called “in vitro” since it occurs in a test tube while “in vivo” signifies occurring in a living cell.

DNA Replication in Cells (in vivo) DNA replication is the copying of DNA It typically takes a cell just a few hours to copy all of its DNA DNA replication is semi-conservative (i.e. one strand of the DNA is used as the template for the growth of a new DNA strand) This process occurs with very few errors (on average there is one error per 1 billion nucleotides copied) More than a dozen enzymes and proteins participate in DNA replication

Polymerase Chain Reaction - PCR PCR amplifies DNA Can makes billions of copies from 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 PCR was invented in the 1984

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 Put all reagents into a PCR tube and into Thermal Cycler 3 Steps Denaturation – 95oC Break the DNA ladder down the middle to create two strands, a 5’ to 3’ strand and a 3’ to 5’ strand Also called melting or heat denaturation Annealing – 60oC Bind each primer to its appropriate strand 5’ primer to the 5’ to 3’ strand 3’ primer to the 3’ to 5’ strand Extension – 72oC Copy each strand DNA polymerase

How PCR Works Temperature Protocol Initial Melt: 94ºC for 2 minutes Denaturation: 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 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 https://www.youtube.com/watch?v=iQsu3Kz9NYo

Gel Electrophoresis of DNA Purpose - Gel electrophoresis detects the presence of DNA in a sample How - 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 Can be used to 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 placed in a gel It is forced, by an electrical current through the gel The gel, microscopically, looks like a sponge filled with small holes The phosphate group in DNA is negatively charged so it moves towards a positive electrode Shorter fragments are able to pass through and move farther along the gel 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 DNA fragments are observed in the gel using a special dye The number of nucleotides are then estimated by comparing it to a known sample of DNA fragments, a ladder, which is run through the gel at the same time

How Gel Electrophoresis of DNA Works Materials Needed Sample of DNA fragments Known sample of DNA fragments DNA ladder Gel Agarose Blue Loading Dye to visualize the movement of the sample as it travels through the gel So know when to stop so sample doesn’t just run off the end of the gel Dye to visualize DNA after it has traveled to its final spot in the gel Syber® Safe Buffer – to carry the electrical current

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

Ladder Well 1 Well 2 Well 3 Well 4 Well 5 Well 6 Well 7 Well 8 Well 9 Well 10 Well 11 Well 12 Ladder