Name: genomic dna extraction methods
Uploaded: 2021-04-07T12:11:59.000Z
Duration: 1 min 24 s
Channel: JoVE

K12 Standards

north-carolina-k-12-science-standard-course-of-study

North Carolina K-12 Science Standard Course of Study

9-12

Biology

heredity-inheritance-and-variation-of-traits

Heredity- Inheritance and Variation of Traits

LS.Bio.8.1

DNA Isolation

DNAは、組換えDNA技術などの多くのアプリケーションにおいて、細胞から分離し、正確な位置で切断する必要があります。
細胞の種類によってDNA抽出方法が異なりますが、標準的なステップは細胞溶解、タンパク質除去、DNA回収の3つです。
真核細胞の内部では、DNAは核内に詰め込まれています。したがって、DNAを単離するには、細胞膜と核膜の両方を破裂させる必要があります。
このステップは、粉砕または超音波処理によって細胞を分解することによって機械的に、または細胞膜の一部を溶解するために界面活性剤および酵素を使用することによって化学的に行うことができる。
細胞内容物が放出されると、破片は遠心分離によって可溶性成分から分離されます。回収された上清には、核酸と水溶性タンパク質が含まれています。
タンパク質を除去するために、プロテイナーゼK、ペプチダーゼ、リゾチームなどの酵素を上清に添加してペプチド結合を切断します。
DNAは、アルコールと酢酸ナトリウムなどの塩を添加することにより、沈殿することにより上清から回収されます。
単離された沈殿物は、水または緩衝液に溶解されます。

<ol>
	<li>Goldenberger, Daniel, Inge Perschil, Michael Ritzler, and Martin Altwegg. 「SDSとプロテイナーゼKを使用したシンプルな」ユニバーサルDNA抽出手順は、直接PCR増幅に適合します。」 Genome Research 4, no. 6 (1995): 368-370.</li>
	<li>「組換えプラスミドDNAをスクリーニングするための迅速なアルカリ抽出手順」 Nucleic acids research 7, no. 6 (1979): 1513-1523.</li>
	<li>Oakes, Christopher C., Sophie La Salle, Jacquetta M. Trasler, and Bernard Robaire. "Restriction digestion and real-time PCR (qAMP)" DNA Methylation, pp. 271-280.ヒューマナプレス、2009年。</li>
</ol>

dnaの単離

DNA単離プロトコルは、さらなる処理に必要なDNAの種類と品質に応じて、高速で簡単な場合もあれば、複雑で時間がかかる場合もあります。 たとえば、プラスミドDNAの抽出は、単離中にgDNAかプラスミドDNAを分離するための適切な溶解方法が必要であるため、ゲノムDNA抽出よりも少し複雑です。ただし、高収量の高品質DNAサンプルを必要とする長距離DNAシークエンシングなどの特定のアプリケーションでは、ゲノムDNA抽出であっても特定のプロトコルに従う必要があります。

ゲノムDNA抽出法の種類

ゲノムDNA抽出法の主な目的は、タンパク質、RNA、およびその他の細胞内容物から gDNA を分離することです。 それには4つの基本的なステップが含まれます。 - 1. 機械的または化学薬品を使用して細胞構造を破壊し、細胞溶解物を取得します。 2. 処理中のDNAの分解から保護します。3. 可溶性DNAを細胞破片から分離します。4. 精製DNAを溶出します。

ほとんどのゲノムDNA単離プロトコルは、溶液ベースまたは固相抽出法のいずれかです。 溶液ベースの方法は、他の細胞物質からDNAを分離するための沈殿および遠心分離のステップに依存し、その後、細胞タンパク質から可溶性DNAを分離するために有機抽出または「塩析」が続きます。 最終的なDNA沈殿はエタノールを使用して行われます。対照的に、固相抽出法では、シリカまたはセルロースマトリックスなどの固体支持体を使用してDNAを結合し、その後洗浄して固体支持体からDNAを溶出します。 これには、結合したDNAを他の細胞成分から分離するための遠心分離、真空、または磁気法が含まれます。

gDNA抽出法の選択は、サンプルの種類、一度に処理するサンプルの数、およびDNAの下流のアプリケーションによって異なります。

DNAの単離

DNA単離プロトコルは、さらに必要なDNAの種類と品質に応じて、迅速かつ簡単な場合もあれば、複雑で時間がかかる場合もあります。

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

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 extraction methods. Solution-based methods use precipitation and centrifugation to separate DNA from other cellular material. They may also use organic extraction or salting out to remove proteins from the soluble DNA. The final DNA is then precipitated with ethanol.

Solid-phase extraction methods use a solid support, such as silica or cellulose matrices, to bind the DNA. The sample is then washed so other cellular components are removed. Next, the DNA is eluted from the solid support. These methods may use centrifugation, vacuum, or magnetic steps to separate the bound DNA during the process.

The best DNA extraction method depends on the sample type, how many samples must be processed, and the next step for the DNA. Plasmid DNA extraction is usually more complex than genomic DNA extraction because the lysis method must separate plasmid DNA from genomic DNA during isolation. In some cases, such as long-range DNA sequencing, specific protocols are needed to obtain enough high-quality DNA.

Genomic DNA Extraction Methods

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 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

Complementary DNA, or cDNA, is made from an RNA template. It is a DNA copy of RNA, so it can help students study gene expression in a lab setting. cDNA is useful when scientists want to work with genes after they have been turned on in a cell. Because it is copied from RNA, it reflects which genes were active at the time the RNA was collected. This process links RNA and DNA in a simple way. It also gives students a clear view of how genetic information can be copied for analysis.

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: 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...

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 genomic DNA is separated from proteins, RNA, and cell debris using solution-based and solid-phase extraction methods for high-quality samples.

Genomic DNA Extraction Methods | 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。

Tennessee Academic Standards

High School

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

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

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

What is Meiosis?

Meiosis is the process by which diploid cells divide to produce haploid daughter cells. In humans, each diploid cell contains 46 chromosomes, half from ...

Meiosis I

Meiosis II

Meiosis vs. Mitosis

Crossing Over

Crossing over is the exchange of genetic information between homologous chromosomes during prophase I of meiosis I. Genetic recombination gives rise to ...

Nondisjunction

Nondisjunction is the failure of homologous chromosomes or sister chromatids to separate correctly and move to the opposite poles of the cells. This ...

Spermatogenesis

Spermatogenesis is the process by which haploid sperm cells are produced in the male testes. It starts with stem cells located close to the outer rim of ...

Oogenesis

In human women, oogenesis produces one mature egg cell or ovum for every precursor cell that enters meiosis. This process differs in two unique ways from ...

Fertilization

During fertilization, an egg and sperm cell fuse to create a new diploid structure. In humans, the process occurs once the egg has been released from the ...

Cleavage and Blastulation

After a large-single-celled zygote is produced via fertilization, the process of cleavage occurs while zygotes travel through the uterine tube. Cleavage ...

Gastrulation

Gastrulation establishes the three primary tissues of an embryo: the ectoderm, mesoderm, and endoderm. This developmental process relies on a series of ...

Neurulation

Neurulation is the embryological process which forms the precursors of the central nervous system and occurs after gastrulation has established the three ...

Cell Migration

Cell migration, the process by which cells move from one location to another, is essential for the proper development and viability of organisms ...

Determination

During embryogenesis, cells become progressively committed to different fates through a two-step process: specification followed by determination. ...

Use models to explain how DNA is passed from parents to offspring through the processes of meiosis and fertilization in sexual reproduction.

LS.Bio.6.1

Mutations

Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations ...

genome-size-and-the-evolution-of-new-genes

Genome Size and the Evolution of New Genes

types-of-genetic-transfer-between-organisms

Types of Genetic Transfer Between Organisms

Base-pairing and DNA Repair

The DNA Replication Fork

Lagging Strand Synthesis

The Replisome

Proofreading

Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template ...

Replication in Prokaryotes

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the ...

Replication in Eukaryotes

In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before ...

Overview of DNA Repair

Base Excision Repair

Nucleotide Excision Repair

DNA Distortion and Damage
Cells are regularly exposed to mutagens&#8212;factors in the environment that can damage DNA and generate mutations. UV ...

Mismatch Repair

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the ...

Fixing Double-strand Breaks

Homologous Recombination

Gene Conversion

Restarting Stalled Replication Forks

Construct an explanation to summarize how inheritable genetic variations may result from: new genetic combinations in meiosis, mutations during replication, or mutations caused by environmental factors.

LS.Bio.6.2

An Overview of Genetic Analysis

An organism&#8217;s physical traits, or phenotype, are a product of its genotype, which is the combination of alleles (gene variants) inherited from its ...

Genetic Crosses

To dissect genetic processes or create organisms with novel suites of traits, scientists can perform genetic crosses, or the purposeful mating of two ...

Punnett Squares

Monohybrid Crosses

Dihybrid Crosses

Trihybrid Crosses

Trihybrid Crosses
Some of Mendel&#8217;s crosses examined three pairs of contrasting characteristics. Such a cross is called a trihybrid cross. A ...

Law of Independent Assortment

Chi-square Analysis

The chi-square test is a statistical hypothesis test. It is used to check whether there is a significant difference between an expected value and an ...

Pedigree Analysis

Multiple Allele Traits

Incomplete Dominance

Gregor Mendel&#39;s work (1822 - 1884) was primarily focused on pea plants. Through his initial experiments, he determined that every gene in a diploid ...

Lethal Alleles

Agouti: A Lethal Allele
Lucien Cu&#233;not discovered lethal alleles in 1905 while studying the inheritance of coat color in mice. The agouti gene is ...

Polygenic Traits

background-and-environment-affect-phenotype

Background and Environment Affect Phenotype

Although the genetic makeup of an organism plays a major role in determining the phenotype, there are also several environmental factors, such as ...

Use mathematics and computational thinking to predict the variation and distribution of expressed traits based on: Mendelian inheritance, co-dominance, incomplete dominance, multiple alleles, and sex-linked inheritance.

LS.Bio.7.1

Overview

A pedigree is a diagram displaying a family&#x2019;s history of a trait. Analyzing pedigrees can reveal (1) whether a trait is dominant or ...

Probability Laws

Overview

The probability of inheriting a trait can be calculated using the sum and product rules. The sum rule is used to calculate the probability of ...

The Concept of Multiple Allelism

Multiple allelism describes genes that exist in three or more allelic forms. Although diploid organisms, like humans, ...

Analyze and interpret data to explain how polygenic traits result in a wide range of phenotypes.

LS.Bio.7.2

Pleiotropy

Pleiotropy is the phenomenon in which a single gene impacts multiple, seemingly unrelated phenotypic traits. For example, defects in the SOX10 gene cause ...

Nature and Nurture

Many human characteristics, like height, are shaped by both nature&mdash;in other words, by our genes&mdash;and by nurture, or our environment. For ...

Construct an explanation to summarize how traits result from interactions of genetic factors (multiple genes and/or alleles) and environmental factors.

LS.Bio.7.3

PCR: The Polymerase Chain Reaction

The polymerase chain reaction, or PCR, is a technique used to amplify DNA through thermocycling &#8211; cyles of temperature changes at fixed time ...

DNA Gel Electrophoresis

DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of ...

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further ...

Restriction Enzymes

Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called ...

DNA Agarose Gel Electrophoresis

Agarose gel electrophoresis is a laboratory technique commonly used to separate DNA fragments by size. However, it can also be used to isolate and purify ...

dna-isolation-and-restriction-enzyme-analysis

DNA Isolation and Restriction Enzyme Analysis

The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our ...

Preparation of Solutions and Materials
ExpandNOTE: For this lab to run smoothly, it is recommended to not exceed 12 samples per laboratory class.
First ...

DNA Isolation
ExpandNOTE: In this experiment you will perform DNA isolation under two experimental conditions: one using a buffer containing the detergent ...

Analyze and interpret data to compare DNA samples.

Understand genetic mechanisms for variation.

Genomic DNA Extraction Methods

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