本期內(nèi)容是 實(shí)驗(yàn)九：聚合酶鏈反應(yīng) PCR，實(shí)驗(yàn)?zāi)M請(qǐng)看下一期。本部分內(nèi)容來自 University of California, Berkeley - UC Berkeley Extension, 虛擬實(shí)驗(yàn)的內(nèi)容來自 Labster. 本部分內(nèi)容均不會(huì)標(biāo)記為為原創(chuàng)，但由于是UP主購買的課程，因此不接受非授權(quán)的轉(zhuǎn)載，謝謝您的理解。

每一個(gè)生物基礎(chǔ)實(shí)驗(yàn)均會(huì)分為三部分：第一部分為實(shí)驗(yàn)的生物理論；第二部分為實(shí)驗(yàn)的指導(dǎo)手冊(cè)；第三部分為 Labster 的虛擬實(shí)驗(yàn)?zāi)M。第一部分的基本信息由 Ying Liu, Ph.D. 提供，第二部分的實(shí)驗(yàn)手冊(cè)來自 Labster, 第三部分的實(shí)驗(yàn)?zāi)M過程由UP主操作。

Virtual Lab Manual 9 -?Polymerase Chain Reaction

Synopsis

In the Polymerase Chain Reaction (PCR) simulation you will be thrown right into a crime scene where a murder has taken place. To investigate the crime scene your first task is to collect blood samples in the hope that the murderer has left traces of their DNA.

Analyze DNA

After sampling you will go to the lab to isolate and Analyze the sample of DNA you collected. By using a PCR kit, a thermocycler, and the purified DNA from the crime scene, it is up to you to mix the correct reagents and perform the PCR experiment.

See the structure of DNA and its replication up close

A 3D animation will show the PCR experiment at the molecular level, illustrating the structure of DNA and its replication. Quiz questions will be asked throughout the experimental process, as well as at specific steps of the PCR itself.

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Identify the murderer

In the PCR simulation, you will collect your sample and other prepared samples from the suspects on a gel, and then compare the patterns that emerge.

Will you be able to identify the murderer?

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Learning Objectives

At the end of this simulation, you will be able to…

●?Explain the function of DNA polymerase in DNA replication and synthesis

●?Perform a PCR experiment using DNA from a blood sample as the template

●?Carry out a gel electrophoresis that separates DNA according to its size

●?Interpret the unique signature of the human genome and the use of tandem repeated regions (TRR) in DNA profiling

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Techniques in Lab

●?Polymerase Chain Reaction (PCR)

●?Gel electrophoresis

●?DNA profiling

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Theory

DNA

DNA (Deoxyribonucleic acid) is the hereditary material of all living organisms, including humans.

DNA is a double helix made of two complementary strands. The information stored in DNA consists of instructions for building and maintaining all the cells of an organism.

The human genome consists of approximately 3.2 billion base pairs. Because of the enormous size of DNA, eukaryotes have developed an efficient?DNA packaging?system.

DNA is found in the nucleus, chloroplasts, and mitochondria of eukaryotes. In prokaryotes, the DNA is not enclosed in a membranous envelope.

DNA fingerprinting

DNA profiling is a way of Analyzing differences among individuals on the?DNA?level. This method is also called DNA fingerprinting, because similar to having unique fingerprints, we also have unique DNA profiles. Large amounts of our DNA are the same in every person, but some regions, called Variable Number Tandem Repeats?(VNTRs), consist of long stretches of small repeated sequences. The number of repeats in any of these sequences differs among individuals due to mutations accumulated through generations. The unique combination of these variable regions makes up the DNA profile. To identify the DNA profile of an individual, we must look at the DNA in these particular regions. The most common approach to do so is to perform an?Short Tandem Repeat (STR) Analysis.

PCR

PCR is a method used to prepare billions of copies of specific?DNA?sequences, i.e, to amplify a DNA sample. It is often necessary to have a larger number of copies of a specific DNA sequence than that found in a typical sample for further DNA Analysis (for example, for?DNA fingerprinting?or?genotyping).

The PCR reaction is highly specific, meaning that it will only produce copies of a desired sequence from the template (sample) DNA. This specificity is ensured by the?primers, which are designed to be complementary and anneal to specific regions on each side of the DNA region of interest (target region).

Gel electrophoresis

Gel electrophoresis is a method to separate charged macromolecules (DNA, RNA, or proteins) of different sizes and to estimate their length.

Because nucleic acids are negatively charged ions at neutral or basic pH in an aqueous environment, this technique is often used to separate DNA or RNA molecules. This is necessary, for example, in the case of?DNA profiling?or to study?RNA integrity.

Gel electrophoresis is often used to separate PCR amplified DNA fragments. The process is also useful to isolate and?extract?DNA fragments of a specific size.

In the virtual lab we use the E-gel machine to perform gel electrophoresis (see image below).

Primers

Primers are short fragments of DNA or RNA used to start?DNA synthesis?by a?DNA polymerase. They are typically 18-25 nucleotides in length and will bind (anneal) to a complementary region of a single-stranded DNA, called the template strand. They mark the beginning of the DNA synthesis to the DNA polymerase. When a primer is bound, the polymerase can also bind to the DNA at the 3' end of the primer and copies the DNA template strand.

DNA polymerase

DNA polymerases are enzymes that are responsible for DNA synthesis in living organisms. These enzymes are able to form a new DNA strand from a template strand by adding nucleotides to the 3 ' end of the primer that is bound to the template DNA .

Nucleotides

Nucleotides are the units on which nucleic acids are built. They are made up of a sugar molecule (deoxyribose in DNA, ribose in RNA), a phosphate group, and a nitrogen-containing base. The sugar molecule is bound to both the phosphate group and the base. Four types of nucleotides can be found in DNA according to the base that is bound to the sugar molecule. The four bases are A (adenine), T (thymine), C (cytosine) and G (guanine).

Taq polymerase

DNA polymerases?are the enzymes responsible for DNA synthesis in living organisms, and therefore they are designed to be functional at physiological conditions. Hence, the DNA polymerase from most organisms would not work well in a?PCR reaction?because most polymerases degrade at 90oC. If such a polymerase was to be used for PCR, it would be necessary to add new DNA polymerase after each cycle. Luckily, a thermostable DNA polymerase was discovered by Thomas Brock: the Taq polymerase. This DNA polymerase was isolated from the bacterium?Thermus aquaticus?found in the hot springs at Yellowstone National Park. The Taq polymerase is active at temperatures up to 95oC. This ensures that multiple cycles of PCR can be performed in a single continuous event, and no additional polymerase is required.

DNA ladder

A nucleic acid "ladder" or molecular weight standard sample is a mix of DNA or RNA fragments with known lengths. It is used as a scale for determining the lengths of unknown nucleic acid fragments when performing?gel electrophoresis experiments. The size of the fragments is determined by running a gel with the ladder in a well next to the samples with unknown lengths. The bands showing from the DNA ladder have predetermined lengths such as "100 bp", "500 bp" and others. If a band from an unknown sample has run the same distance in the gel as the 500 bp band, it can be assumed that the unknown fragment has a length close or equal to 500.

A gel electrophoresis is usually run with a "ladder" in one of the wells. Fragment lengths are given in base pairs. In this image also the concentration of the particular fragment in the "ladder" is displayed in ng/10uL.

Sterile technique

Sterile technique is used to ensure a "clean" lab environment. It is essential to ensure the reliability of?experimental results.

Sterile practices are especially important when working with microorganisms. A single spore or tiny bacterium can overgrow your whole medium and destroy your experiment.

The following steps are used to keep laboratory work sterile:

●?Laboratory doors and windows are kept closed to prevent air currents, preventing surface microorganisms becoming airborne.

●?The wire loop and glass spreader are?sterilized?before and after use with a Bunsen burner to prevent the introduction of unwanted microorganisms.

●?Lids from bottles and tubes are held when removed, and not placed on the bench during material transfer from one bottle or tube to another.

●?The neck of a bottle or tube must be immediately heated using the Bunsen burner so that any air movement is outward.

●?The bottle or tube are opened for the minimum time possible, and while open, all work is performed close to the Bunsen burner flame.

●?Media and equipment are sterilized to prevent the growth of unwanted microorganisms.

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