Name: dna extraction, quantification, and cutting
Uploaded: 2019-01-31T09:26:45.000Z
Duration: 4 min 46 s
Channel: JoVE

K12 Standards

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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-and-restriction-enzyme-analysis

DNA Isolation and Restriction Enzyme Analysis

DNA 提取是从细胞中分离和纯化 DNA 的过程。首先，细胞被裂解——即打破细胞结构——通常通过物理破碎与化学试剂处理相结合的方式实现，例如使用能够溶解细胞膜和核膜的去污剂。十二烷基硫酸钠（SDS）是一种常用的去污剂，其作用原理是溶解构成这些膜结构的蛋白质和脂质。随后，细胞内容物便可自由释放到周围溶液中。

接下来，必须将DNA与其他存在的分子分离开。向反应体系中加入蛋白酶K，可降解肽键并消化污染的蛋白质。然后向样品中加入盐，以稳定DNA骨架中带负电荷的磷酸基团；随后加入预冷的醇类试剂，使DNA从溶液中沉淀析出。白色沉淀物通过离心收集，沉淀将沉降至离心管底部。经洗涤并重新悬浮于缓冲液后，所提取的DNA即可用于科研或生物技术应用。

某些生物技术应用，例如可识别个体或多个个体特异性 DNA 新模式的 DNA 指纹图谱技术，涉及限制性内切酶的使用。限制性内切酶是一类能够与 DNA 相互作用并识别特定序列的分子。一旦识别出其特异性位点，便会切割 DNA，导致 DNA 链被切割成一个或多个线性片段。如果提取的 DNA 是质粒（一种通常存在于细菌中的环状 DNA 分子），则任何切割都会使 DNA 形成线性片段或多个片段。不同的限制性内切酶识别不同的 DNA 序列，因此联合使用这些酶可产生不同的特异性片段。

在DNA指纹分析中，我们可以使用一种称为DNA电泳或凝胶电泳的技术来检测这些片段。制备凝胶时，将琼脂糖粉末与缓冲液混合并加热至完全溶解。然后向温热的混合液中加入核酸染料，再将该溶液倒入制胶模具中。插入梳子以形成加样孔。待凝胶凝固后，将其转移至盛有缓冲液的电泳槽中，并移去梳子。将已知长度的带染料DNA片段混合物（即DNA分子量标记）加入其中一个加样孔，待测的染色DNA样品则加入其余加样孔中。将电泳槽连接至电源，通电后，DNA核苷酸中带负电荷的磷酸基团会在电场作用下通过凝胶向阳极（正极）迁移。较小的DNA片段迁移较快，而较大的片段则因迁移阻力较大而移动较慢。

当电泳结束后，将凝胶暴露于紫外光下，以观察DNA样本中的核苷酸染色情况。可根据其相对于DNA标准条带的位置来确认其存在。由于不同序列的DNA在不同位置具有酶切位点，因此会产生独特的条带模式，即“指纹图谱”，可用于区分个体或不同的DNA变异特征。

在本实验中，您将使用含有SDS和不含SDS的缓冲液进行DNA提取，以评估去污剂在DNA分离中的重要性，随后用不同的限制性内切酶消化质粒DNA，以分析所产生的DNA图谱。

dna 提取与限制性内切酶分析

DNA作为所有生物遗传分子的发现，带来了巨大的科学与医学突破，并显著加深了我们对自身及其他生物的理解。DNA的分离与分析是过去一个世纪中众多科技进步的基础性首要步骤，从基因功能的鉴定，到农业和法医学的革命性发展，均依赖于此。

<h4>从细胞中分离DNA</h4>

DNA 提取是一个相对简单的流程，需要经过几个但至关重要的步骤：细胞收集、裂解、蛋白质降解，最后是 DNA 沉淀。通常，DNA 可从少量组织中获取，例如植物的一片叶子、动物的毛囊，或通过简单的生理盐水溶液在口腔内轻易脱落的上皮细胞。由于 DNA 在真核细胞中被局限于细胞核或线粒体内，科学家首先需破坏细胞膜和核膜以暴露 DNA，这一过程通过裂解步骤实现。然而，一旦 DNA 暴露，就可能受到重金属离子和极端 pH 值的降解作用。因此，使用含有去污剂的缓冲液不仅可溶解细胞膜，还能保护 DNA 免受溶液中降解性成分的影响。此外，DNA 是一种异常长的分子，其在细胞核内通过紧密缠绕在称为组蛋白的特殊蛋白质周围而高度压缩。因此，需加入一种名为蛋白酶 K（Proteinase K）的酶，该酶可降解蛋白质的肽键，从而将 DNA 与组蛋白及其他蛋白质分离。接下来，科学家利用 DNA 的化学性质将其从其余细胞提取物中分离出来。由于核酸具有带负电荷的结构，氯化钠溶液中的 Na+ 离子会结合并嵌入到缠结的 DNA 链中。一旦 DNA 聚集成团，即可用肉眼观察到。DNA 在酒精中不溶，并在低温下迅速沉淀，因此科学家会加入冷酒精以促使 DNA 沉淀。最后，沉淀的 DNA 可溶解并保存于去离子水或温和缓冲液中，这些溶液不含任何可降解核酸的酶，直至后续使用。

<h4>定量 DNA</h4>

使用 DNA 的反应通常需要高纯度和精确量的 DNA。因此，在 DNA 分离后，科研人员会使用分光光度计来测定样品中 DNA 的含量以及 DNA 的纯度。分光光度计通过测量穿过样品的光束强度随波长的变化来工作。核酸在 260 nm 处吸收光，而蛋白质在 280 nm 处吸收光。通过测量 260 nm 处的光束强度，科研人员可以确定 DNA 的浓度。此外，通过检测 260 nm 和 280 nm 处的光强度，可以判断 DNA 的纯度。在纯 DNA 样品中，260/280 nm 的比值应接近 2。

<h4>限制性内切酶</h4>

对于多种分析，例如克隆、测序以及DNA片段和基因组的作图，需要使用限制性内切酶在特定序列处切割分离得到的DNA。这些酶由细菌产生，作为抵御外源病毒DNA入侵的先天免疫机制，其功能类似于分子剪刀。因此，当限制性内切酶识别出病毒DNA后，便会通过在其特异性识别的核苷酸序列处进行切割，将DNA分解成片段。这些核苷酸序列通常长度为6至12个核苷酸，且通常具有回文结构，即在3’-5’和5’-3’方向上的核苷酸序列相同。例如，限制性内切酶EcoRI在序列为3’...GAATTC…-5’的位点切割DNA，该序列在5’-3’方向上读取时也相同。与EcoRI类似，已有数百种酶分别对应不同的回文序列。DNA经这些酶切割后，科研人员可将DNA样品加入电泳槽中的凝胶，并施加电流。DNA带有负电荷，因此DNA片段会向阳极迁移。然而，凝胶中的孔隙会使较大的DNA片段迁移速度慢于较小的DNA片段，从而实现按大小分离。电泳完成后，可通过特异性结合DNA分子的染料对DNA片段进行可视化观察。

使用限制性内切酶切割 DNA，并通过凝胶电泳分离 DNA 片段，可使研究人员相对于标准 DNA Marker 确定未知片段的大小。随后可进一步分离这些目标片段，并将其连接至质粒载体中，用于基因克隆。这些方法极大地推动了基因工程和实验室分析的发展。通过 DNA 指纹分析，科学家能够对基因组进行测序，从而更深入地理解生物体的运作机制，同时也能识别和表征特定基因1。

DNA 的分离与分析在直接影响日常生活的事件中也具有实际应用价值。例如，法医科学家 routinely 分析 DNA 样本，为刑事和民事案件提供证据2。同样，医疗保健人员通过提取和分析 DNA 来进行亲子鉴定和遗传病检测。近年来，DNA 族源分析试剂盒日益流行，使用户能够确定自身的遗传谱系、寻找亲属，甚至发现自身对多种健康状况的遗传易感性3。最后，随着生物技术和基因工程的进步，个性化医学在临床研究人员中逐渐受到重视，通过利用个体的遗传数据开发治疗方案，为有效治疗患者且避免严重副作用铺平了道路4。

<h4>参考文献</h4>

<ol class="references">
	<li>Chambers, GK, 等 动物学中的DNA指纹技术：过去、现在与未来。Investig Genet. 2014年，第5卷，3 doi: 10.1186/2041-2223-5-3。</li>
	<li>Roewer, L. 法医学中的DNA指纹技术：过去、现在与未来。Investig Genet. . 2013年，第4卷，22 doi: 10.1186/2041-2223-4-22。</li>
	<li>Kirkpatrick, BE 和 Rashkin, MD. 祖源检测与遗传咨询实践。J Gen Counseling. 2016年，第26卷，第1期（6-20）。</li>
	<li>Hamburg, MA 和 Collins, FS. 通往个体化医学之路。N Engl J Med . 2010年；第363卷，301-304。</li>
</ol>

DNA 提取与限制性内切酶分析

DNA被揭示为所有生物体的遗传分子，已带来巨大的科学与医学突破，并显著推动了我们对……的理解

Education

教育

JoVE Lab Manual

生物学

Genetics

遗传学

DNA 分离和限制性内切酶分析

dna extraction, quantification, and cutting

DNA extraction, quantification, and cutting are key steps in many biology labs. Scientists use these methods to study genes, prepare DNA for testing, and compare fragments for analysis.

DNA isolation starts with a small sample, such as a leaf, hair follicles, or epithelial cells from the mouth collected with a saline solution. The first steps are cell harvesting, lysis, protein degradation, and DNA precipitation. Lysis breaks open the cell and nuclear membranes so the DNA can be released from the nucleus or mitochondria in eukaryotic cells. A detergent-containing buffer helps dissolve membranes and also protects DNA from degrading conditions such as heavy metal ions and extreme pH.

After the DNA is exposed, Proteinase K is added. This enzyme breaks peptide bonds in proteins and helps separate DNA from histones and other proteins. The DNA is then mixed with sodium chloride, and Na+ ions help the tangled DNA strands clump together because nucleic acids carry negative charges. Cold alcohol is added next, since DNA does not dissolve in alcohol and will precipitate at low temperatures. The DNA can then be dissolved in deionized water or a mild buffer for storage, as long as the solution is free of enzymes that could break it down.

Once DNA is isolated, scientists often measure how much DNA they have and how pure it is. A spectrophotometer does this by sending light through the sample at specific wavelengths. DNA absorbs light at 260 nm, while proteins absorb light at 280 nm. The 260 nm reading shows DNA concentration, and the 260/280 ratio helps estimate purity. In a sample of pure DNA, that ratio should approach 2.

For cloning, sequencing, and mapping DNA fragments or genomes, scientists often need to cut DNA at specific sites. They use restriction enzymes, which are bacterial proteins that act like molecular scissors against invading viral DNA. These enzymes recognize short nucleotide sequences, often six to twelve bases long, and the sequences are usually palindromic, meaning they read the same in both directions. EcoRI is one example and cuts at the sequence GAATTC.

After cutting, the DNA fragments are loaded into a gel and separated by electrophoresis. DNA is negatively charged, so it moves toward the anode when an electric current is applied. Smaller fragments travel faster through the gel pores than larger ones, so the pieces separate by size. Dyes that bind to DNA make the fragments visible, and researchers compare them with a standard ladder to estimate fragment size. The fragments can then be isolated and ligated into a plasmid vector for genetic cloning.

These methods support many modern applications. Scientists use DNA profiling to sequence genomes and to identify and study genes. DNA analysis also helps in forensics for criminal and civil cases, in paternity testing, and in checks for genetic diseases. Ancestry kits use DNA to help people trace lineage, find relatives, and learn about possible health traits. These tools also support personalized medicine, where researchers use a person’s genetic data to design treatments with fewer harmful side effects.

DNA Extraction, Quantification, and Cutting

dna-extraction-quantification-and-cutting

genetics of organisms

Mendelian Traits and Exceptions

Mendelian genetics explains how traits move from parents to offspring. Gregor Mendel studied pea plants in 1865 and showed that many visible traits are controlled by two alleles, or gene versions, with one inherited from each parent. He also found that some alleles are dominant and others are recessive. Mendel’s work was not widely accepted until 1900, and it took about 30 more years for Mendelian inheritance to fit into evolutionary theory. One reason for the delay was that real breeding...

dna isolation and restriction enzyme analysis

DNA extraction, quantification, and cutting are key steps in many biology labs. Scientists use these methods to study genes, prepare DNA for testing, and compare fragments for analysis. DNA isolation starts with a small sample, such as a leaf, hair follicles, or epithelial cells from the mouth collected with a saline solution. The first steps are cell harvesting, lysis, protein degradation, and DNA precipitation. Lysis breaks open the cell and nuclear membranes so the DNA can be released from...

bacterial transformation

Plasmid DNA Transfer in Bacteria

Plasmid DNA transfer in bacteria helped launch modern genetic engineering. The idea traces back to Frederick Griffith’s work on pneumonia-causing Streptococcus pneumoniae in the early 20th century. He studied a rough, non-virulent R strain and a smooth, virulent S strain with a protective polysaccharide capsule. When he injected mice with heat-killed bacteria of either strain, the mice survived. But when heat-killed S-strain bacteria were mixed with live R-strain bacteria, the mice died, and...

genetics

Learn how DNA is isolated, measured with a spectrophotometer, cut with restriction enzymes, and separated by gel electrophoresis for analysis.

DNA Extraction, Quantification, and Cutting | JoVE K12

通过这段 2 分钟的教师指南，准备教授 DNA 提取与限制性内切酶分析。涵盖 JoVE Lab Manual 上 Biology 实验的关键准备步骤。

Biology 中的 DNA 提取与限制性内切酶分析 教师准备 - 2.5K+ 观看次数 | JoVE Lab Manual

在这段 13 分钟的学生实验流程视频中，逐步学习 DNA 提取与限制性内切酶分析 的操作步骤。属于 JoVE Lab Manual 上的 Biology 系列。

Biology 中的 DNA 提取与限制性内切酶分析 学生实验流程 - 4.0K+ 观看次数 | JoVE Lab Manual

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

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 ...

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.

Transgenic Organisms

Reproductive Cloning

CRISPR

Synthetic Biology

Synthetic biology is an interdisciplinary science that involves using principles from disciplines such as engineering, molecular biology, cell biology, ...

Ribosome Profiling

Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences ...

Transgenic Plants

Recombinant DNA technology called transgenesis is often used to add a foreign gene or remove a detrimental gene from an organism. Such genetically ...

Gene Therapy

biofuels-producing-ethanol-from-cellulosic-material

Biofuels: Producing Ethanol from Cellulosic Material

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
In this experiment, cellulosic material (such as corn stalks, leaves, ...

Testing For Genetically Modified Foods

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
Genetic modification of foods has been a controversial issue due to debated ...

Obtain and communicate information that summarizes the impact of biotechnology applications on the individual, society, and the environment, including agriculture and medicine.

LS.Bio.8.2

Understand genetic mechanisms for variation.

DNA Extraction, Quantification, and Cutting

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