DNA Extraction, PCR, and Gel Electrophoresis PDF

DNA EXTRACTION, PCR AND GEL ELECTROPHORESIS DNA Extraction: Significance  To obtain DNA in a relatively purified form which can be used for further investigations such as:  PCR (polymerase chain reaction)  Sequencing  Genetic Manipulation  Electrophoresis...

DNA EXTRACTION, PCR AND GEL ELECTROPHORESIS DNA Extraction: Significance  To obtain DNA in a relatively purified form which can be used for further investigations such as:  PCR (polymerase chain reaction)  Sequencing  Genetic Manipulation  Electrophoresis  Finger Printing  Cloning Confirming the presence and quality of the DNA  For further lab work, it is important to know the concentration and quality of the DNA.  spectrophotometer can be used to determine the concentration and purity of DNA in a sample.  gel electrophoresis can be used to show the presence of DNA in the sample and give an indication of its quality. Confirming Presence of DNA: gel electrophoresis  Presence of DNA can be confirmed by electrophoresing on an agarose gel containing ethidium bromide, or another fluorescent dye that reacts with the DNA, and checking under UV light. DNA Extraction  Breaking cells open to release the DNA  Separating DNA from proteins and other cellular debris  Precipitating the DNA with an alcohol  Cleaning the DNA  Confirming the presence and quality of the DNA Polymerase chain reaction (PCR)  Primer - a short sequence of nucleotides that  A technique used to create several copies of provides a starting point for DNA synthesis a certain DNA segment. usually around 20 nucleotides in length.  Developed in 1983 by Kary Mullis.  Two primers are used in each PCR reaction, and they are designed so that they flank the  Can generate millions of copies of a small target region (region that should be copied). segment of DNA.  called "molecular photocopying  Goal: to make enough of the target DNA region that it can be analyzed or used in some other way. PCR requirements needed 1. DNA Template– The DNA of interest from The steps of PCR the sample. 2. DNA Polymerase– Taq Polymerase 1. Denaturation (96 °C)  thermostable enzyme and does not 2. Annealing (55-65°C) denature at very high temperatures. 3. Extension (72 °C) 3. Oligonucleotide Primers- short stretches of single-stranded DNA 4. Deoxyribonucleotide triphosphate– Nucleotide solution mix containing adenine (A), thymidine (T), cytosine (C), and guanine (G) used to build duplicate DNA strands 5. Buffer System–provide optimum conditions for DNA denaturation and renaturation.  Important for fidelity, polymerase activity, and stability. Taq polymerase  PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates.  The DNA polymerase typically used in PCR is called Taq polymerase (Thermus aquaticus)  T. aquaticus lives in hot springs and hydrothermal vents.  very heat-stable and is most active around 70 °C (a temperature at which a human or E. coli DNA polymerase would be nonfunctional) 1. Denaturation (96 °C)  This heat-stability makes Taq polymerase  The sample containing the template ideal for PCR DNA are heated to 94-96⁰C  to separate, or denature, the double PCR primers stranded DNA strands  Taq polymerase can only make DNA if it's  To provides single-stranded given a primer template for the next step.  - takes 2-4hrs hours, depending on the length 2. Annealing of the DNA region being copied.  The reaction temperature is lowered to - produce millions/billions of copy of the 54-60℃ for around 20- 40 seconds. target DNA  Primers bind to their complementary sequences on the template DNA.  Primers are single-strand sequences of DNA or RNA 20 to 30 Bp.  starting point for the synthesis of DNA.  two primers needed- a forward primer and a reverse primer. 3. Extension  Temperature is raised to 72-80℃.  Addition of complementary bases to the Gel Electrophoresis 3’ end of the primer by the Taq  A technique used to separate DNA, RNA, & polymerase enzyme. protein based on their size and charge.  It attaches to the primer and adds DNA bases to the single strand.  Electrophoresis involves running a current through a gel containing the molecules of  This elongates the DNA in the 5’ to 3’ interest. direction.  Based on their size and charge, the  The DNA polymerase adds about molecules will travel through the gel at 1000bp/minute different speeds, allowing them to be  As a result, a double-stranded DNA separated from one another. molecule is obtained.  Gels for DNA separation are often made out of a polysaccharide called agarose.  Gel has pocket-like indentations called wells, which are where the DNA samples will be placed.  Electrophoresis involves running a current through a gel containing the molecules of interest Gel electrophoresis Procedure - The cycle repeats 25-40 times Requirements Needed:   DNA sample  A well-defined “line” of DNA on a gel is called  Current– run DNA from negative to positive a band. under the influence of electrical pulses  Each band contains a large number of DNA  Agarose gel– provide space or work as a fragments of the same size vehicle to run DNA under the current  By comparing the bands in a sample to the Buffer– a medium that facilitates easy DNA ladder, we can determine their  migration approximate sizes.  Dyes– track and monitor the migration of DNA. 1. Preparing the samples for running 2. Preparing the agarose gel solution 3. Casting the gel 4. Loading the gel 5. Electrophoresis 6. Visualizing the DNA  A scientist purified samples of DNA from several puppies. She expected to determine if there are any twin puppies in the group.  The image depicts an agarose gel in which a set of DNA size reference standards (lane 1) were electrophoresed alongside the Visualizing the DNA fragments scientist's experimental DNA fragments (lane  the gel will be stained with a dye that is 2-7). fluorescent in color.  Based on the results of this analysis, are any  The dye will bind to the DNA fragments. of these puppies likely to be identical  The fragments are put on the ultraviolet transilluminator to see some bright bands which are the stained DNA. DNA Ladder  The DNA ladder is a standardsized molecular marker or fragments of known lengths used to determine the size of PCR amplicons.  Stable at room temperature and ready for immediate gel loading.  50bp to 10kb and contain loading dye for simple migration observation. Analyzing and Interpreting (Agarose) Gel Electrophoresis Results  Some factors that affects the results of agarose electrophoresis o The concentration of gel o Re-use of chemicals and solutions o Unpure DNA samples o Concentration of buffer o The concentration of agarose gel