Name: dna extraction, quantification, and cutting
Uploaded: 2019-01-31T09:26:45.000Z
Duration: 4 min 46 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-and-restriction-enzyme-analysis

DNA Isolation and Restriction Enzyme Analysis

DNA 提取是从细胞中去除和纯化 DNA。首先，细胞被裂解 - 破裂 - 通常是通过物理破坏和化学品处理的组合，例如溶解细胞和核膜的洗涤剂。SDS 或十二烷基硫酸钠是一种常用的去污剂，其作用是溶解构成这些膜的蛋白质和脂质。然后内容物可以自由漂浮到周围的溶液中。

接下来，必须将 DNA 与存在的其他分子分离。添加到反应中的蛋白酶 K 会分解肽键并消化污染的蛋白质。然后将盐添加到样品中，以稳定 DNA 骨架中带负电荷的磷酸基团，并在加入冰冷的酒精后，将 DNA 从溶液中沉淀出来。通过在离心机中旋转收集白色沉淀物，使其沉降到试管底部。在缓冲溶液中洗涤并重悬后，提取的 DNA 最终可用于研究或生物技术应用。

一些生物技术应用，如 DNA 指纹图谱，可以识别一个或多个个体特有的 DNA 中的新模式，涉及限制性内切酶的使用。限制性内切酶是与 DNA 相互作用并识别特定序列的分子。一旦确定了它们的特定部位，他们就会切割 DNA。这将导致发束被切割成一个或多个线性块。如果提取的 DNA 是质粒，即细菌中最常见的环状 DNA 片段，则任何切割都会导致 DNA 形成线性片段或片段。不同的限制性内切酶识别不同的 DNA 序列，因此使用它们的组合可以产生不同的片段。

在 DNA 指纹图谱中，我们可以使用一种称为 DNA 或凝胶电泳的技术来检查这些片段。为了制备凝胶，将琼脂糖粉与缓冲液混合并加热直至溶解。然后将核苷酸染色剂添加到温热的混合物中，然后将该溶液倒入铸模中。插入梳子以形成孔。凝固后，将凝胶转移至装满缓冲液的凝胶盒中，并取出凝胶梳。将已知长度的染色 DNA 片段的参比混合物 DNA 分子量标准品加入一个孔中，并将染色的目标 DNA 样品上样到其余孔中。盒子连接到电源，打开电源会诱导 DNA 核苷酸中带负电荷的磷酸基团通过凝胶向阳极（正端）迁移。较小的碎片比较大的碎片移动得更快，而较大的碎片则很难迁移。

电泳完成后，将凝胶暴露在紫外光下，以观察 DNA 样品中的核苷酸染色。可以根据它们与梯形带的相对位置来确认它们的存在。由于具有不同序列的 DNA 在不同位置具有切割位点，因此可以产生新的条带模式或指纹，可用于区分个体或变体 DNA 图谱。

在本实验中，您将使用含和不含 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，然后使用不同的限制性内切酶消化质粒 DNA，以检查所得的 DNA 谱。

dna 分离和限制性内切酶分析

DNA 是所有生物体的遗传分子，这一发现带来了巨大的科学和医学突破，并显着增强了我们对自身和其他生物体的理解。DNA 分离和分析是上个世纪许多进步的基本第一步;从基因功能的鉴定到农业和法医学的革命。

<h4>从细胞中分离 DNA</h4>

DNA 分离是一个相当简单的程序，只需要几个但必不可少的步骤:细胞收获、裂解、蛋白质降解，最后是 DNA 沉淀。通常，可以从少量组织中收集 DNA，例如植物的单片叶子、动物的毛囊或上皮细胞，这些细胞可以很容易地用简单的盐水溶液在口腔内脱落。由于 DNA 在真核细胞内的细胞核或线粒体内被分隔开，科学家首先打破细胞和核膜以暴露 DNA，这是通过裂解步骤完成的。然而，一旦 DNA 暴露，它就会被重金属离子和极端的 pH 值降解。因此，含有去污剂的缓冲溶液不仅可以溶解细胞膜，还可以保护 DNA 免受溶液中元素的降解。此外，DNA 是一种异常长的分子，通过紧密堆积在称为组蛋白的特殊蛋白质周围而浓缩在细胞核内。因此，添加了一种称为蛋白酶 K 的酶，它可以降解蛋白质的肽键，以将 DNA 与组蛋白和其他蛋白质分离。接下来，科学家利用其化学特性将 DNA 与细胞提取物的其余部分分离。由于核酸的带负电荷的结构，氯化钠溶液中的 Na+ 离子与缠结的 DNA 链结合并整合到其中。一旦 DNA 结块，就可以用肉眼观察到。DNA 不溶于酒精，在低温下容易沉淀，因此科学家添加冷醇来沉淀 DNA。最后，沉淀的 DNA 可以溶解并储存在去离子水或温和的缓冲液中，不含任何可降解核酸的酶，直到使用。

<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，科学家使用分光光度计来定量样品中 DNA 的量，以及样品中 DNA 的纯度。分光光度计测量穿过样品的光束强度与其波长的关系。核酸吸收 260 nm 的光，而蛋白质吸收 280 nm 的光。通过测量 260 nm 光束的强度，科学家可以确定 DNA 浓度。此外，通过评估 260 nm 和 280 nm 处的光强度，可以确定 DNA 含量的纯度。在纯 DNA 样品中，260/280 nm 比率应接近 2。

<h4>限制性内切酶</h4>

对于许多分析，例如克隆、测序和绘制 DNA 片段和基因组图谱，需要使用限制性内切酶在特定序列处切割分离的 DNA。这些酶由细菌产生，是针对入侵病毒 DNA 的先天免疫机制，起到分子剪刀的作用。因此，在限制性内切酶识别出病毒 DNA 后，它们开始通过将其切割成它们识别的某些核苷酸序列的片段来将其分解。这些核苷酸序列通常有 6 到 12 个核苷酸长，通常是回文的，这意味着它们在 3'-5' 和 5'-3' 方向上具有相同的核苷酸序列。例如，限制性内切酶 EcoRI 在序列 3'...GAATTC...-5'，在 5'-3' 方向上的读数相同。与 EcoRI 一样，有数百种酶对应于许多不同的回文序列。一旦 DNA 被这些酶切割，科学家就可以将 DNA 加载到电泳室中的凝胶上，并通过凝胶传递电流。DNA 带有负电荷，导致 DNA 片段向阳极迁移。然而，相对于较小的 DNA 序列，凝胶的孔会减慢较大的 DNA 片段的速度，因此这些片段根据其大小分离。电泳完成后，可以借助与 DNA 分子特异性结合的染料观察 DNA 片段。

使用限制性内切酶切割 DNA 并通过电泳分离 DNA 片段，使研究人员能够识别未知片段相对于标准分子量标准的大小。然后可以进一步分离这些目标片段并将其连接到质粒载体中以进行基因克隆。这些方法极大地改进了基因工程和实验室分析。通过 DNA 分析，科学家能够对基因组进行测序以进一步了解生物体的工作原理，并且还能够识别和表征某些基因1。

DNA 分离和分析对于直接影响日常生活的事件也很实用。例如，法医科学家通常会分析 DNA 样本，以便为刑事和民事案件提供证据2。同样，医护人员提取和分析 DNA 以检查亲子关系和遗传疾病。最近，DNA 血统分析试剂盒开始流行起来，它允许用户确定他们的遗传谱系、寻找亲属，甚至发现他们对各种健康状况的遗传倾向3。最后，随着生物技术和基因工程的进步，个性化医疗在临床研究人员中越来越受欢迎，通过使用个人的基因数据来开发治疗方法，为有效治疗患者而没有有害副作用铺平了道路4。

<h4>引用</h4>

<ol class="references">
	<li>钱伯斯、GK 等人。动物学中的 DNA 指纹识别:过去、现在、未来。Investig Genet. 2014， Vol. 5， 3 doi: 10.1186/2041-2223-5-3.</li>
	<li>罗沃 L.法医中的 DNA 指纹识别:过去、现在、未来。调查基因。 .2013， Vol. 4， 22 doi: 10.1186/2041-2223-4-22.</li>
	<li>Kirkpatrick，BE 和 Rashkin，医学博士。血统检测和遗传咨询的实践。J Gen 咨询。 2016 年，第 26 卷，1 （6-20）。</li>
	<li>马萨诸塞州汉堡和科林斯，FS。个性化医疗之路。N Engl J Med . 2010;，第 363 卷，301-304。</li>
</ol>

DNA 分离和限制性内切酶分析

DNA 作为所有生物体的遗传分子的揭示导致了巨大的科学和医学突破，并显着增强了我们的......

Education

教育

JoVE Lab Manual

生物学

Genetics

遗传学

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 分离和限制性内切酶分析。涵盖 JoVE Lab Manual 上 Biology 实验的关键准备步骤。

Biology 中的 DNA 分离和限制性内切酶分析 教师准备 - 14K+ 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 分离和限制性内切酶分析 的操作步骤。属于 JoVE Lab Manual 上的 Biology 系列。

Biology 中的 DNA 分离和限制性内切酶分析 学生实验流程 - 33K+ 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

Description

Transcript

Tags

More videos by Standards