Name: pcr: dna amplification for genetics labs
Uploaded: 2021-04-07T12:12:00.000Z
Duration: 1 min 32 s
Channel: JoVE

K12 Standards

Tennessee Academic Standards

High School

Biology

links-among-engineering-technology-science-and-society

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

PCR - Polymerase Chain Reaction

PCRの目的は、ポリメラーゼ連鎖反応によって 遺伝子配列を増幅することです。この例では、目的の遺伝子が精製DNAから 増幅されます。増幅を行うためには、ポリメラーゼが新しいＤＮＡを合成するために必要となります。目的の遺伝子と相同性を共有する，一本鎖DNAの短い断片であるプライマーも 必要となります。最後に、デオキシヌクレオチド三リン酸(dNTP)という単一ヌクレオチドを用いて新しい鎖を作ります。PCRの最初のステップは、DNAを変性させる 混合物を加熱することです。次に反応物を冷却し、そしてプライマーをそれらの相同領域にアニーリングさせます。一度結合すると、反応は ポリメラーゼにとって最適な温度に加熱される。次いでポリメラーゼは プライマーDNA複合体を認識し，溶液中のdNTPを用いて 新しい鎖の合成を開始します。次いで反応物を加熱し、このように 2回目のサイクルが進行します。3回目のサイクルのあとには，遺伝子のコピーは合計8個です。4回目の後は16個です。5回目の後は32個です。コピー数は指数関数的に増え続けます。30サイクル後には、10億以上のコピーがあります。

「研究技術をシンプルに:ポリメラーゼ連鎖反応(PCR)」 The Journal of Investigative Dermatology 133, no. 3 (March 2013): e6.[<a href="https://doi.org/10.1038/jid.2013.1" target="__blank">ソース</a>]

「バイオテクノロジー革命:PCRと発現遺伝子をクローンするための逆転写酵素の使用」 Nature Education 1、no. 1(2008):94。[<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">ソース</a>]

pcr - ポリメラーゼ連鎖反応

<h4>概要</h4>
 
 ポリメラーゼ連鎖反応(PCR)は、DNAの断片をコピーするために広く使われている技術です。指数関数的な増幅により、PCRはわずか数時間で数百万から数十億のDNAコピーを作り出すことができます。PCR反応では、耐熱性のDNAポリメラーゼ酵素が、サーモサイクラーと呼ばれる自動機械の中で、一連の温度変化を通して元のDNAを増幅します。
 
 <h4>PCRは分子生物学に革命をもたらした汎用性の高い手法</h4>
 
 1983年にカリー・マリス(Kary Mullis)がPCRを開発し、1993年にノーベル化学賞を受賞しました。PCRは、比較的早く、安価で、正確にDNA配列をコピーできるため、分子クローニング、遺伝子変異誘発、病原体の検出、遺伝子発現解析、DNAの定量と配列決定、遺伝病の診断など、さまざまな用途で非常に重要なツールとなりました。 
 
 <h4>PCRの反応サイクル</h4>
 
 PCRは、細胞内で起こる自然なDNA複製過程を模倣しています。反応混合物には、コピーされるべき鋳型DNA配列、プライマーと呼ばれる一対の短いDNA分子、デオキシヌクレオチド三リン酸（dNTPs）と呼ばれる遊離のDNA構成要素、および特殊なDNAポリメラーゼ酵素が含まれます。
 
 PCRは高温で一連の作業を行うため、そのような温度でも機能するDNAポリメラーゼ酵素が必要です。最も一般的なDNAポリメラーゼは、最初にポリメラーゼが分離された細菌であるThermus aquaticusにちなんで名付けられたTaqポリメラーゼです。DNAポリメラーゼは、DNA分子をゼロ（de novo）から合成することはできません。その代わりに、DNAポリメラーゼはプライマーと呼ばれる相補的な塩基対に結合する短いDNA分子を介してDNAテンプレートに結合します。プライマーは、DNAポリメラーゼが新しいdNTPを結合できる遊離の3’ヒドロキシ基を提供します。PCRでは、DNA分子の各ヌクレオチドに対応して、dATP、dCTP、dGTP、dTTPの4種類のdNTPが使用されます。 
 
 各PCRサイクルは、「変性」、「アニーリング」、「DNA合成」の3つのステップで構成されています。
 
 <ol>
 <li>変性。PCRサイクルは、反応混合物を高温に加熱し、DNAの二重らせんを2本に解離することで開始されます。この解離プロセスは、通常、90°C­­–100°C前後の温度で行われます。</li>
 <li>アニーリング。反応混合物を急速に冷却し（通常50°C–65°C）、2つのプライマーが鋳型DNA鎖上の相補的な配列に結合できるようにします。 </li>
 <li>DNA合成（プライマー伸長）：反応混合物を再度加熱し、今度はDNAポリメラーゼが鋳型鎖の塩基と対になるdNTPを加えてプライマーを伸長できる温度（通常60°C–75°C）にします。</li>
 </ol>
 
 一般的なPCRでは、これら3つのステップをサーモサイクラーで20～40サイクル繰り返します。各サイクルでDNA分子の数が2倍になるため、指数関数的にDNAが増幅されていきます。
 
 <h4>PCRの限界</h4>
 
 もし科学者がゲノムの特定の配列を増幅したい場合、科学者は適切なプライマーを設計するために、ターゲットDNAの配列の少なくとも一部を知っていなければなりません。また、部分的に類似したDNA配列にプライマーが非特異的にアニーリングしてしまい、非標的DNAが増幅されてしまうという問題もあります。この問題は、反応条件を最適化することで抑制できます。高感度の検出法であるPCRは、汚染にも弱く、微量の汚染DNAが混入しただけで、誤解を招く結果となります。PCRに使用されるDNAポリメラーゼは、エラーを起こしやすいです。最初の数サイクルで突然変異が起こると、増幅されたDNAのほとんどがその変異を持つことになります。

PCR - ポリメラーゼ連鎖反応

概要

ポリメラーゼ連鎖反応(PCR)は、DNAのセグメントをコピーするために広く使用されている技術です。指数関数的な増幅により、PCRは...

Education

JoVE Core

Molecular Biology

分子生物学

Unit 2

ユニット2

Studying DNA and RNA

DNAとRNAの研究

KEY TERMS AND CONCEPTS

主要用語と概念

pcr: dna amplification for genetics labs

PCR is a DNA amplification method used in genetics labs. PCR stands for polymerase chain reaction. It copies a specific DNA segment so scientists can study small amounts of genetic material.

The process uses repeated heating and cooling steps. These steps let the DNA strands separate, primers bind to the target region, and a DNA polymerase build new strands. With each cycle, the target DNA can increase in amount.

PCR is a core technique in molecular biology and many school-level biology topics. It helps students connect DNA structure with laboratory methods used to analyze genetic information.

PCR: DNA Amplification for Genetics Labs

recombinant dna

Recombinant DNA and Gene Cloning

Recombinant DNA and gene cloning are tools used to combine DNA from different sources. Scientists use them to isolate a gene, join it to a carrier DNA molecule, and make copies of that DNA. A plasmid is one common carrier used in this process. A plasmid is a small ring of DNA found in bacteria, and it can carry the inserted gene into a host cell. Once inside the cell, the recombinant DNA can be copied as the cell grows. This approach is important in biotechnology and genetic research. It...

dna isolation

Genomic DNA Extraction Methods

Genomic DNA extraction methods separate DNA from proteins, RNA, and other cell material. The process usually follows four main steps. First, the cell structure is disrupted with mechanical or chemical methods to make a cell lysate. Then the DNA is protected from damage during processing. After that, the soluble DNA is separated from cell debris. Finally, the purified DNA is eluted, or washed out, for later use. DNA isolation can follow two main approaches: solution-based methods or solid-phase...

dna agarose gel electrophoresis

Purifying DNA Bands from Agarose Gels

Agarose gel electrophoresis can be used to purify DNA bands after separation by size. In gel extraction, the DNA fragment of interest is cut from the gel and cleaned before later use. This is especially useful in cloning, where pure DNA is needed before ligation, the joining step that links DNA pieces together. In cloning experiments, both the insert and vector DNA fragments are first cut with restriction endonucleases. These fragments often contain enzymes, salts, and other contaminants. Such...

labeling dna probes

DNA Probe Labels for Detecting Targets

DNA probes are short DNA fragments with a reporter tag attached so they can be detected or purified. They bind to matching nucleic acid sequences by complementary base-pairing. That binding helps researchers recover or identify specific regions of DNA. Several reporter tags are commonly used to label DNA probes. These include radioisotopes, fluorophores, and small molecule binding partners such as biotin or digoxigenin. The label choice depends on the probe’s intended use and on the source of...

southern blot

Southern Blot for DNA Detection

Southern blotting is a DNA detection method used after agarose gel electrophoresis separates DNA fragments by size. A DNA ladder with fragments of known length is run beside the sample so the fragment size can be estimated. The blot adds another step when scientists need to confirm the sequence identity of the DNA. First, the target DNA is cut into smaller pieces with a restriction endonuclease and run on an agarose gel. The DNA is then denatured, which means the double strands are separated...

dna microarrays

Gene Chips for Comparing Cell Activity

DNA microarrays are gene chips used to compare gene activity in different conditions. They are high-throughput assays, which means they can analyze large amounts of data at once. They are also relatively inexpensive and can be automated, making them useful for genome-wide studies. These chips are built on glass or silica slides. Probe molecules are covalently attached to the surface through surface functionalization. In many cases, the slides are prepared by chemisorption of silanes to silica...

complementary dna

Making cDNA from RNA Templates

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fish - fluorescent in-situ hybridization

FISH for Chromosome and Pathogen Detection

FISH is a DNA and RNA detection method used in cytogenetics and medical testing. FISH stands for fluorescence in situ hybridization. It uses labeled probes that bind to complementary DNA or RNA sequences on a chromosome or inside a cell. The method was developed in the early 1980s and became widely used because it can show specific genetic targets with high detail. In the past, probes were made by cloning or by reverse transcription of a DNA template. Today, probe oligonucleotides can also be...

pcr - polymerase chain reaction

PCR is a DNA amplification method used in genetics labs. PCR stands for polymerase chain reaction. It copies a specific DNA segment so scientists can study small amounts of genetic material. The process uses repeated heating and cooling steps. These steps let the DNA strands separate, primers bind to the target region, and a DNA polymerase build new strands. With each cycle, the target DNA can increase in amount. PCR is a core technique in molecular biology and many school-level biology...

real time rt-pcr

Real-Time RT-PCR for Gene Expression Tracking

Real-time RT-PCR tracks gene expression by measuring how much target DNA is made during PCR. It starts with mRNA, which is converted into complementary DNA, or cDNA, by an enzyme called reverse transcriptase. The cDNA is then amplified by PCR so the target sequence can be measured. This method can be done in one tube as a single-step reaction or in two separate steps. During amplification, the amount of product is followed in real time with fluorescent dyes or sequence-specific probes linked...

race - rapid amplification of cdna ends

Finding cDNA Ends with RACE PCR

Rapid Amplification of cDNA Ends, or RACE, helps scientists capture the full-length cDNA from an mRNA sequence. It works by linking a known internal region to the unknown sequence at either the 5' end or the 3' end. The method is widely used when the missing end of a gene needs to be identified. RACE uses a gene-specific primer that binds to the known cDNA region. A hybrid primer then adds a set anchor sequence to the unknown end of the cDNA. After that, PCR amplifies the DNA segment between...

sanger sequencing

How Sanger Sequencing Reads DNA Fragments

Sanger sequencing reads DNA fragments by separating pieces of different lengths and decoding the order of bases. It is a key method in biology and is often used to study a cloned DNA fragment, a gene mutation, or even much larger genomes. Fredrick Sanger and his collaborators developed the chain-termination method in 1977. The method works by producing a mixture of DNA fragments with variable sizes. These fragments are then separated by capillary gel electrophoresis, which sorts them by length.

next-generation sequencing

DNA Sequencing Methods and Uses

DNA sequencing methods can read millions of DNA fragments at the same time. Next-generation sequencing made this process much faster and much less expensive than older methods. The first human genome project cost $2.7 billion and took 15 years. It was completed in 2003 after major international cooperation among research teams and funding agencies. To prepare DNA for next-generation sequencing, pure genomic DNA is first broken into smaller pieces. These pieces are used to build a sequencing...

rna-seq

RNA-Seq Workflow and Data Analysis

RNA sequencing, or RNA-Seq, is a high-throughput method for studying the transcriptome of a cell. The transcriptome is the full set of RNA molecules in a sample. Transcriptomics helps scientists understand how genomes work, how organisms develop, and how some diseases occur. Before RNA-Seq, researchers often used microarray methods and Sanger sequencing to study RNA. Microarrays had limited coverage and depended on already knowing the genome sequence. Sanger sequencing was low-throughput,...

genome annotation and assembly

How Genome Assemblies Are Updated

Genome assembly and genome annotation are key steps in genome analysis. Genome assembly puts DNA sequencing data back in the correct order to make a close copy of the original genome. Genome annotation then identifies functional parts on that assembled genome. Assembling a genome is not simple. In human populations, gene copy number can vary, and repeated DNA sequences can make the process harder. Even so, the physical location of human genes stays constant. Bacterial genomes can be more...

restriction enzymes

DNA Cutting Sites and Bacterial Defense

Restriction enzymes are bacterial proteins that cut DNA at specific sequence sites. They recognize palindromic DNA sequences, also called inverted repeats, and cut the DNA in a sequence-specific way. These sites are often found in regions with important functions, such as the origin of replication, gene operator sites, and transcription termination signals. Bacteria protect their own DNA by methylating these same sites. Methylation means adding chemical marks that keep the restriction enzyme...

maxam-gilbert sequencing

Chemical Cleavage DNA Sequencing

Chemical cleavage DNA sequencing uses specific chemicals to cut DNA at known sites. Allan Maxam and Walter Gilbert described this method in the same year that the Sanger sequencing method was discovered. It reads the DNA sequence by breaking purified DNA into fragments and then studying the fragment pattern. The method depends on chemicals that cleave DNA at specific bases. The resulting fragments are separated by electrophoresis, which is a way to sort DNA pieces by size using an electric...

key terms and concepts

Learn how PCR amplifies a specific DNA segment through heating, cooling, primers, and DNA polymerase in genetics and molecular biology labs.

PCR: DNA Amplification for Genetics Labs | JoVE K12

molecular biology

JoVE Core 分子生物学は、明確で簡潔なアニメーション ビデオ レッスンを通じて細胞の分子プロセスを紹介します。さらに、科学者の実際のビデオでは、これらの概念の応用が現代の研究室での実際の実験を通じて実証されています。

Florida Department of Education

Life Science

SC.912.L.16

SC.912.L.16.12

Texas Essential Knowledge and Skills

BIO.7

BIO.7.D

myJoVE Corporation

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BIO1.ETS2.3

PCR: DNA Amplification for Genetics Labs

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