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- Timea Gerczei Fernandez and Scott Pattison
- Ball State UniversitydoorDeGruyter
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4.1Learning objective
In this lab you perform site-directed mutagenesis using the QuickChange Mutagenesis Kit (Stratagene). You will learn how polymerases work and how to amplify DNA using the polymerase chain reaction (PCR).
4.2Mini project flowchart
The bold block in the flowchart below emphasizes the role of the current experiment in the mini project.
4.3Overview of the nucleic acid structure
Before learning how polymerases work and the requirements for successful PCR amplification, you should review a few things about nucleic acid structure.NucleinezurenThey are polymers of nucleotides. Nucleotides are held together by phosphodiester bonds in the nucleic acid. Figure 4.1 shows the structure of a nucleotide.nucleotidesThey consist of a sugar group: ribose (RNA) or deoxyribose (DNA); a heterocyclic aromatic group: the nucleobase (A, C, G, T or U) and a phosphate group. When referring to functional groups within the sugar group, we use the apostrophe symbol; for example, the second hydroxyl group of the ribose nucleotide is called the 2'-OH group.
As we mentioned earlier, nucleotides are held together by the phosphodiester bond in the nucleic acid chain. Hephosphodiester linkageIt is formed by two phosphate ester bonds: each bond is formed between an OH group of ribose or deoxyribose and an OH group of phosphate. Since phosphoric acid is a moderately strong acid, the phosphodiester bond deprotonates under physiological conditions, giving nucleic acids a negative charge (Figure 4.2).
have nucleic acid chainspolaritysuch as protein chains. Nucleic acid polymers begin with the 5' phosphate of the first nucleotide and end with the 3' OH group of the last nucleotide in the chain. Therefore, the nucleic acid chain has a precise direction; runs from the 5' end to the 3' end, just as protein strands run from the N-terminus to the C-terminus. In a double-stranded nucleic acid such as DNA (our genetic material), a DNA polymer runs from 5' to 3' and it is calledtop strandand the other goes from 3' to 5' in the direction mentionedlower strand. These two strands of DNA are complementary to each other: adenine (A) versus thymine (T) and cytosine (C) versus guanine (G) to ensure proper Watson-Crick base pairing between the strands. In other words, the two DNA strands of this double-stranded DNA (dsDNA) areAdditionallyeach other. Complement means that the two threads are complementary to each other and the direction of the warp is opposite: the top thread runs from 5' to 3' and the bobbin thread runs from 3' to 5'.
5’ AGGCCATTGGA 3’
3’ TCCGGTAACCT 5’
4.4How do polymerases work?
Polymerases synthesize nucleic acids using a nucleic acid template. The sequence of the newly synthesized nucleic acid will beAdditionallyfrom the template series. During nucleic acid synthesis, the 3'-OH group of the growing nucleotide chain acts as a nucleophile to attack the phosphorus of the incoming nucleotide, apyrophosphate(PAGESi) leaves and the phosphodiester bond is formed. This means that the newly synthesized DNA strand grows in the 5' to 3' direction (Figure 4.3).
To properly position the nucleophile to attack divalent metal ions (usually Mg2+) are required for successful DNA synthesis.
Polymerase mechanism in a nutshell:
– Polymerases synthesize DNA using a template that is the complement of the newly synthesized DNA.
– To synthesize DNA, polymerases need an OH group that acts as a nucleophile. This OH comes from the growing nucleic acid chain. Recognize that the reaction is a nucleophilic substitution.
– The leaving group is pyrophosphate (PPi), a high-energy molecule that splits into two inorganic phosphates. This reaction is catalyzed by the enzyme inorganic phosphatase.live.
– Polymerization is energetically favorable, because two high-energy anhydride bonds (one in the incoming nucleotide triphosphate and the other in pyrophosphate) are broken and a stable bond (ester bond connecting the nucleotides) is formed.
– To initiate DNA synthesis, DNA polymerases have aprimerto provide the required OH group as a nucleophile.
– Polymerases travel from 3' to 5' on the bottom strand of the dsDNA template as they synthesize the growing strand in the 5' to 3' direction.
4.5Polymerase chain reaction (PCR) in practice
To synthesize DNA in the lab, we need to perform PCR amplification of the DNA of interest using a plasmid vector or genomic DNA as a template. For a successful PCR amplification, we need to perform three steps between 25 and 30 times. The PCR amplification steps are as follows:
- Separation of the dsDNA template or strand separation.
- Annealing of the primers with the template DNA (they form Watson-Crick base pairs) to initiate DNA synthesis.
- DNA synthesis catalyzed by a polymerase.
Figure 4.5 shows a graphical representation of a PCR cycle.
![1.4: Perform site-directed mutagenesis (7)](https://i0.wp.com/bio.libretexts.org/lt-bio-18921" "1.4: Perform site-directed mutagenesis (7)")
4.6Why was PCR not widely available until the 1980s?
PCR amplification required two scientific breakthroughs. (1) The scientist had to invent a programmable machine that can go through the PCR cycle temperatures multiple times. The PCR machine can change the temperature accurately and quickly; within seconds the temperature can go from 95 ºC to 68 ºC. (2) The scientists had to find a DNA polymerase that could survive the first step of the PCR cycle: incubation at 95ºC. This step is necessary to separate the dsDNA strands. Since polymerases are proteins, most bacterial or eukaryotic polymerases would not survive this incubation (think about what happens when you boil an egg). The scientists focused on organisms that live under extreme conditions: in volcanic springs with high temperatures. Most of these organisms belong to the third kingdom of life.you bow. The polymerases of these organisms have evolved to be active at 60-80 ºC, so incubation at 95 ºC for a few minutes will not harm them.TaqThe polymerase has a half-life of 2 hours. at 95 °C whilefunnyTurbopolymerase has a half-life of 19 hours. at 95°C. In addition, the polymerase used for PCR must have high fidelity; this means that the polymerase rarely incorporates an incorrect nucleotide into the synthesized DNA (it has a low error rate).TaqPolymerase has a 16% error rate on a 1 kilobase DNA sequenceUf turboPolymerase has a 2.6% error rate on the same sequence. Our PCR amplification uses an Archaeal DNA polymerase.mad pyrokok(funnyturbo).
4.7PCR applications
PCR is used to amplify DNA present in only a few copies by several orders of magnitude. As a result, PCR amplification generates millions of copies of the desired DNA sequence. PCR has revolutionized molecular biology and has a myriad of applications beyond basic science. Some of these apps are highlighted below.
- Specific DNA Isolation:Using primers designed for the gene in question, a particular DNA sequence can be amplified. This DNA can then be inserted into a cloning vector for further study or sequence.
- DNA-kwantificering(quantitative real-time PCR or qRT-PCR). This method estimates the amount of a given nucleic acid sequence present in the sample. Since each PCR cycle theoretically doubles the amount of nucleic acid given, the fewer amplification cycles it takes to generate a detectable amount of DNA, the higher its concentration was in the original sample. This means that gene expression levels can be determined. The method requires simultaneous amplification and detection (in real time). The primers amplify the nucleic acid of interest in the presence of a DNA-specific fluorescent dye. The number of cycles required to obtain detectable fluorescence intensities (CTnumber). This number is inversely proportional to the concentration of the target nucleic acid in the sample.
- Genetic mapping.Using specific primers, million-fold amplification of a specific part of the genome can be achieved. That way, even if only a few copies of the given DNA are available, enough DNA can be generated by PCR for scientific research. This property of PCR has given rise to a large number of applications beyond basic science.DNA profile(genetic fingerprinting) is a technique used by forensic scientists to identify a person involved in a crime or to establish the parent-child relationship between individuals (paternity testing). Although 99.9% of the sequence of the human genome is shared, there are enough differences between each person to allow identification. The most commonly used test procedures today focus on short tandem repeats (STR). These regions of the human genome are highly variable in sequence, meaning that individuals are extremely unlikely to have similar sequences unless they are closely related. Only monozygotic twins, 'identical twins', have the same short tandem repeat sequences.
- PCR at diagnosis.Using specific primers sensitive to a particular pathogen (bacteria or virus), the source of infection can be identified much more quickly by PCR than by culturing samples. Similarly, mapping specific parts of the genome can reveal whether an individual is predisposed to breast cancer or other diseases that are much more treatable if detected at an early stage.
PROCEDURES
The needs for reagents and equipment are calculated by six student teams. A deductible of ~20% is included.
Required equipment/glassware:
- PCR-machine
- Three sets of 20-100 μl, 2-20 μl and 1-10 μl micropipettes
- 6 tubes for PCR
- 6 centrifugebuizen
Necessary reagents
- Pfu Turbo Polymerase Mastermix (Stratagene); 130 µl totaal
- DpnI restriction enzyme (6 μl total)
- Primers to generate each mutant.
- Plasmid DNA containing the ykkCD toxin sensor
To set up the PCR reaction, carefully mix the following reagents, place the tubes in the PCR machine and start the protocol.
- 20 μl 2X Pfu Turbo Mastermix
- 100 ng plasmid DNA (volume depends on DNA concentration)
- 1 µl top primer of 1 µM
- 1 µl 1 µM light primer
- Water to 40 µl
The PCR machine is programmed as follows:
Remove the template DNA (does not contain the mutation)
- Remove the reactions from the PCR machine and centrifuge briefly to ensure that all of the reaction mixture is at the bottom of the tube.
- Add 1 µl of DpnI restriction enzyme to each tube.
- Incubate the reactions for 1 hour in a 37 °C water bath.
- Reactions can be stored at -20°C until use.
Notes for instructors
The experiment in Chapter 4 is designed to perform site-directed mutagenesis in theBacillus subtilisykkCD tetracycline sensor RNA. The minimal modification protocol could be used to perform site-directed mutagenesis on any nucleic acid. Use of a high-fidelity DNA polymerase, such asUf Turbo,It is essential to the success of the experiment, but alternative suppliers or packaging may be used. Primers were ordered from Integrated DNA Technologies and reconstituted in 1 x TE (Tris-EDTA) at a concentration of 1 µM. Plasmid DNA containing the ykkCD sensor RNA is available from the authors upon request. Because this PCR reaction takes approximately two hours, it is helpful to start the lab session by setting up the PCR reactions and lecturing the lab while the reactions are running. This laboratory is extremely suitable for a long research because of the minimal wet laboratory work and the long reaction time. Depending on the time allotted for the lab session, the teaching assistant or instructor may need to delete the PCR reactions.
Pre-laboratory questions for site-directed mutagenesis
Define the following terms.
- nucleotide
- Nucleic acid
- reverse complement
- Below is the bottom strand of a dsDNA. What is the order of the upper chain? Mark the 3' and 5' ends of the top strand sequence.
3' AAGTTCAAGGC 5'
- Calculate how to mix your PCR reaction if your plasmid DNA concentration is 275 ng/µl (use the Protocol section of your booklet). Show your work.
Site-directed mutagenesis
Overview of the laboratory report and distribution of points
- Several sentences defining the purpose of this experiment (2 points).
- Briefly describe the DNA polymerase reaction. Why is it thermodynamically favorable (about 5 sentences; 4 points)?
- Briefly describe the site-directed mutagenesis of Quickchange (5-10 sentences; 5 points).
- Describes the first cycle of a PCR reaction. Be sure to specify the temperature at which each cycle will run. Briefly explain why significant amplification is obtained with PCR (4 points).
- Describes the first cycle of Quickchange PCR. Briefly explain why you get less amplification with Quickchange than with normal PCR (5 points).
- PCR-werkblad (30 pnt.).
PCR-werkblad
DNA structure:
1. (3 pt.) Draw the structure of pdApdCpdT. Label the 5' and 3' ends and circle each of the phosphodiester bonds in this small nucleic acid.
2. (3 points) Show the H-bond pairing between adenine and its complementary base.
3. (3 points) A small molecule of double-stranded DNA is studied. Given the sequence of a DNA strand:
GCTACATTCGGAA
Write down the sequence of the other DNA strand. (Note that sequences are written from the 5' to 3' direction.)
Standard PCR:
4. (3 points) Why is PCR described as a “chain reaction”?
5. (3 points) Explain why two PCR cycles are needed to make the desired DNA product.
6. (3 points) In PCR, a certain amount of DNA polymerase is added at the beginning, which must remain active during all subsequent cycles. This requirement severely limits which DNA polymerases can be used. Why is this so?
QuickChange DNA-synthese:
7. (3 points) Site-directed mutagenesis allows scientists to specify exactly where base changes in DNA will occur (mutagenesis). How does the QuickChange process enable us to achieve “site-directed mutagenesis”?
8. (3 points) Standard PCR allows “amplification” of the DNA product. Does QuickChange allow the same type of “gain”? To explain.
9. (3 points) Standard PCR requires a primer to be attached to the beginning of the DNA template sequence. A QuickChange primer, on the other hand, does not have to bind at the beginning of the template, but can bind anywhere in the sequence. How do you explain this difference?
10. (3 points) Standard PCR can be used to make many copies of a newly isolated gene. QuickChange, on the other hand, is only useful for making base changes to a well-studied DNA sequence. What is different about the QuickChange process that makes such a difference in using it?