Episode 33 recAPs genetic engineering techniques of biotechnology.
Episode 33 recAPs genetic engineering techniques of biotechnology. Electrophoresis separates molecular fragments according to size and charge visually (1:00). PCR amplifies DNA fragments, making thousands of copies from even the smallest sample (2:35). Bacterial transformation introduces foreign DNA into bacterial cells (4:00). DNA sequencing is a part of biotechnology, but typically not working with an entire genome at one time (6:15).
The Question of the Day asks (8:08) “Which cells in the human body do NOT contain nuclear DNA?
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Hi and welcome to the APsolute Recap: Biology Edition. Today’s episode will recap Biotechnology
Lets Zoom out:
Unit 6 - Gene Expression and Regulation
Topic - 6.8
Big Idea: Information Storage and Transmission
Genetic engineering techniques can be used to analyze and manipulate DNA and RNA. You do not need to know specific details of these techniques for the AP exam, but rather a conceptual understanding of how they are applied. The main processes we will recap are electrophoresis, PCR, bacterial transformation and DNA sequencing.
Lets Zoom in:
First up - Electrophoresis. This process separates molecular fragments according to size and charge visually. The experimental set up can look intense - but each part has a purpose. Electrophoresis is run in a gel box that has electrodes at either end and is filled with a salt buffer solution which conducts current. Within the box is a porous gel that contains indented wells, one for each DNA sample.
The DNA samples that were previously treated with specific restriction enzymes are pipetted into the wells nearest the negative electrode. The samples are often dyed and weighted so that they sink into the wells. The current is turned on and the gel typically “runs” for 1-2 hours. Because DNA fragments are negatively charged due to their phosphate group, they migrate away from the well in columns toward the positive electrode according to size. Small fragments of DNA containing fewer base pairs move farther down the gel than larger ones. Once the gel has been stained, you will see the DNA fragments as distinct bands. You will need to measure the bands and graph this data on a logarithmic base pairs scale. Analysis of DNA can be used for forensic identification. This process is often used to identify samples of DNA to a control group (as in crime scene samples to a suspect).
Next, PCR or polymerase chain reaction. This process amplifies DNA fragments, making thousands of copies from even the smallest sample. Amplified DNA fragments can be used to identify organisms (or suspects) and perform phylogenetic analysis. With a little temperature manipulation and the right ingredients in a test tube, PCR can create hundreds of thousands of DNA copies in a few hours.
You are in the lab with your test tube - it contains your small DNA sample to serve as a template, a salt solution with cofactors, RNA primers, free nucleotides and DNA polymerase. Step ones is denaturation at 96 degrees celsius. This separates the double helix so that nitrogenous bases are exposed and accessible. Step two is primer annealing at 55 degrees celsius. This cooling of the test tube allows for RNA primers to complementary bind to each single-stranded DNA template (you know - C to G, A to U). Step three is extension at 72 degrees celsius. A heat tolerant DNA polymerase known as Taq polymerase synthesizes 5’ to 3’ from the RNA primer. And then the process starts all over again! With each heating and cooling cycle, the growth of DNA strands is exponential. The thousands of copies of DNA can now be used in other areas of research and application.
Bacteria have a bad reputation, but many of them are necessary and even helpful for our survival. Bacterial transformation introduces foreign DNA into bacterial cells. I mean, it sounds cool - but why would we do that? Bacteria reproduce very quickly, every 30 minutes for some species. When scientists want to study a specific gene or need many copies of a protein product - bacteria serve as a xerox machine. Insert the right gene, into the right plasmid, and watch the bacteria multiply.
Bacteria have a circular chromosome that can take in smaller DNA plasmids. Big picture of how this works, with an emphasis on temperature, time, and sterilization techniques. First, the plasmid has to be prepared. This involves cutting the sequence open with restriction enzymes, inserting the gene we are studying, and then closing the plasmid with DNA ligase. Restriction enzymes are sequence specific, scanning and splicing the plasmid with either sticky or blunt ends. The plasmid is now a molecule of recombinant DNA, which has been assembled from pieces from multiple sources. Your teacher likely ordered prepared plasmids for you to work with during the bacterial transformation lab.
Next, conditions need to be right for the bacteria to take in the plasmid. This is commonly done by a heat shock (putting microtubes in a warm water bath) for about one minute. This causes some of the bacteria to take up the plasmid. It is common to couple the gene you are studying with another, like antibiotic resistance, to track which bacteria successfully took in the plasmid. Using sterile loops, the bacteria is then plated on agar, sometimes treated with antibiotics or arabinose. After a period of incubation (not too hot, not too cold - the goldi-locks environment for bacterial growth). The bacteria which took in the plasmid will grow and express the new plasmid gene. Tada! The bacteria have turned into little gene copying, protein producing factories for you - the scientist. This technique is a type of DNA cloning. One example of bacterial protein production is with insulin used by diabetics.
The human genome project began on October 1, 1990 and concluded in April of 2003. This was a remarkable feat to decode the As, Ts, Cs, and Gs of of our species. Decode might be a leap - we at least know the order of the letters. Still trying to figure out all the meaning behind them. It's like seeing the cake and having the recipe, but not knowing which step produces the frosting and which step tells you to mix in the flour. DNA sequencing is a part of biotechnology, but typically not working with an entire genome at one time.
A common method of sequencing shorter base pairs is with the Sanger method. In this strategy, the DNA sample is copied in different sized fragments. Each end is marked with a fluorescent chain terminator which allows the sequence to be determined. Basically by overlapping the pieces and finding the patterns. To complete a Sanger sequence you need DNA polymerase, a primer, the DNA template, free nucleotides and Dideoxy nucleotides. Didoxy nucleotides lack a hydroxyl at the 3’ carbon, and as such are unable to covalently bond to another nucletide’s phosphate group. As a result, they terminate the chain. Through a similar heat, cool, heat cycle as PCR - multiple fragments are produced. The data is then run through capillary gel electrophoresis and displayed in a chromatogram. Since the shortests fragments travel farthest, the sequence is determined by reading the peaks of the chromatogram.
To recap….
Biotechnology is pretty cool and involves the manipulation of DNA for research and investigations. Often, DNA samples are first amplified by PCR in order to be used in electrophoresis and transformation. Don’t worry about the specific steps, but focus on how the structure of DNA relates to each process and the practical applications of each.
Today’s Question of the day is about DNA
Question - Which cells in the human body do NOT contain nuclear DNA?