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 extraction is the removal and purification of DNA from cells. First, cells are lysed - broken open - usually though a combination of physical disruption and treatment with chemicals, such a detergent which dissolves the cell and nuclear membranes. SDS or sodium dodecyl sulfate is a commonly use detergent that works by solubilizing the proteins and lipids that make up these membranes. The contents can then float freely into the surrounding solution. 

Next, DNA must be separated from the other molecules present. Proteinase K added to the reaction will break down peptide bonds and digest contaminating proteins. Salt is then added to the sample, which stabilizes the negatively charged phosphate groups in the backbone of the DNA and, after the addition of ice cold alcohol, precipitates the DNA out of solution. The white precipitate is collected by spinning it in a centrifuge where it settles to the bottom of the tube. After washing and resuspending it in a buffer solution, the extracted DNA can finally be used in research or biotechnology applications. 

Some biotechnology applications, like DNA fingerprinting, which can identify novel patterns in DNA specific to an individual or individuals, involve the use of restriction enzymes. Restriction enzymes are molecules which interact with DNA and recognize specific sequences. Once their specific site is identified, they cut the DNA. This will result in the strand being cut into one or more linear pieces. If the DNA extracted was a plasmid, a circular piece of DNA most often found in bacteria, any cuts will result in the DNA forming a linear fragment or fragments. Different restriction enzymes recognize different DNA sequences, so using a combination of these can result in distinct fragments being produced. 

In DNA fingerprinting, we can then examine these fragments using a technique called DNA or gel electrophoresis. To prepare gels, powdered agarose is mixed with a buffer and heated until dissolved. A nucleotide stain is then added to the warm mixture and then this solution is poured into the casting mold. A comb is inserted to form the wells. When solidified, the gel is transferred to a gel box filled with buffer and the comb is removed. A reference mixture of dyed DNA fragments of known lengths, the DNA ladder, is added to one well and the dyed DNA samples of interest are loaded into the remaining wells. The box is connected to a power source and switching the power on induces the migration of the negatively charged phosphate groups in DNA nucleotides through the gel towards the anode, the positive end. Smaller pieces move more quickly than the larger fragments, which migrate with difficulty. 

When the run is complete the gel is exposed to ultra violet light to visualize the nucleotide stain in the DNA samples. Their presence can be confirmed based on their relative location to the ladder bands. Because DNA with different sequences will have cut sites at different locations, this can produce novel band patterns, or fingerprints, which can be used to distinguish individuals or variant DNA profiles. 

In this lab you will perform DNA extractions using a buffer with and without SDS to assess the importance of detergents in DNA isolation and then digest plasmid DNA with different restriction enzymes to examine the resulting DNA profiles.

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 understanding of ourselves and other organisms. DNA isolation and profiling have been the fundamental first steps for many of the advancements in the past century; from identification of gene function, to revolutions of agriculture and forensics.

<h4>Isolating DNA from Cells</h4>

DNA isolation is a fairly simple procedure that requires few but essential steps: cell harvesting, lysis, protein degradation, and finally DNA precipitation. Generally, DNA can be harvested from small amounts of tissue, such as a single leaf of a plant, hair follicles of animals, or epithelial cells that can be easily sloughed off within the mouth with a simple saline solution. Since DNA is compartmentalized inside the nucleus or mitochondria within eukaryotic cells, scientists first break apart the cell and nuclear membrane to expose the DNA, which is done with the lysis step. However, once DNA is exposed, it can be degraded by heavy metal ions and extremes of pH. Therefore, a detergent-containing buffer solution is used to not only dissolve the cell membrane, but also to protect DNA from degrading elements in the solution. Moreover, DNA is an unusually long molecule that is condensed inside the nucleus by packing tightly around special proteins called histones. Therefore, an enzyme called Proteinase K, which degrades peptide bonds of proteins, is added to separate the DNA from histones and other proteins. Next, scientists separate DNA from the rest of the cellular extract by using its chemical properties. Due to the negatively charged structure of nucleic acids, Na+ ions in a sodium chloride solution bind to and integrate into entangled DNA strands. Once the DNA is clumped, it can be observed by the naked eye. DNA is insoluble in alcohol and readily precipitates at low temperatures, thus scientists add cold alcohol to precipitate DNA. Finally, the precipitated DNA can be dissolved and stored in deionized water or a mild buffer, free of any enzymes that can degrade nucleic acids, until it is used.

<h4>Quantifying DNA</h4>

Reactions that use DNA often require high purity and precise amounts of DNA. Therefore, following DNA isolation, scientists use a spectrophotometer to quantify the amount of DNA in their sample, as well as the purity of the DNA in the sample. A spectrophotometer measures the intensity of a light beam passed through a sample as a function of its wavelength. Nucleic acids absorb light at 260 nm, whereas proteins absorb light at 280 nm. By measuring the intensity of a light beam at 260 nm, scientists can determine the DNA concentration. Furthermore, by assessing the light intensity at both 260 nm and 280 nm, the purity of the DNA content can be determined. In a sample of pure DNA, the 260/280 nm ratio should approach the value of 2.

<h4>Restriction Enzymes</h4>

For a number of analyses, such as cloning, sequencing, and mapping DNA fragments and genomes, the isolated DNA needs to be cut at specific sequences using restriction enzymes. These enzymes are produced by bacteria as mechanisms of innate immunity against intruding viral DNA and function as molecular scissors. Hence, after restriction enzymes identify a viral DNA, they start to break it apart by cutting it into segments at certain nucleotide sequences they recognize. These nucleotide sequences are often six to twelve nucleotides long and are generally palindromic, which means that they have the same nucleotide sequence in the 3&#8217;-5&#8217; and 5&#8217;-3&#8217; directions. For instance, the restriction enzyme EcoRI cuts DNA at the sequence 3&#8217;...GAATTC&#8230;-5&#8217;, which reads the same in the 5&#8217;-3&#8217; direction. Like EcoRI, there are hundreds of enzymes that correspond to many distinct palindromic sequences. Once DNA is cut by these enzymes, scientists can load the DNA onto a gel in an electrophoresis chamber and pass electrical current through the gel. DNA carries negative charges, which causes DNA fragments to migrate towards the anode. However, the pores of the gel slow down larger DNA fragments relative to smaller DNA sequences, thus the pieces separate according to their size. After the completion of the electrophoresis, DNA fragments can be visualized with the aid of dyes that specifically bind to DNA molecules.

Cutting DNA with restriction enzymes and separating DNA fragments with electrophoresis allows researchers to identify the sizes of unknown fragments relative to a standard ladder. These fragments of interest can then be further isolated and ligated into a plasmid vector for genetic cloning. Genetic engineering and laboratory analyses have been improved immensely with these methods. With DNA profiling, scientists are able to sequence genomes to further understand the workings of organisms and are also able to identify and characterize certain genes1.

DNA isolation and profiling are also practical for events directly impacting everyday life. For example, forensics scientists routinely analyze DNA samples to provide evidence for criminal and civil cases2. Similarly, healthcare workers extract and analyze DNA to check for paternity and genetic diseases. Recently, DNA ancestry analysis kits became popular by allowing users determine their genetic lineage, find relatives, and even discover their genetic disposition to various health conditions3. Finally, with advancements in biotechnology and genetic engineering, personalized medicine is gaining traction among clinical researchers to develop treatments by using an individual&#8217;s genetic data, paving the way to effectively treat patients without detrimental side effects4.

<h4>References</h4>

<ol class="references">
	<li>Chambers, GK, et al. DNA fingerprinting in zoology: past, present, future. Investig Genet. 2014, Vol. 5, 3 doi: 10.1186/2041-2223-5-3.</li>
	<li>Roewer, L. DNA fingerprinting in forensics: past, present, future. Investig Genet. . 2013, Vol. 4, 22 doi: 10.1186/2041-2223-4-22.</li>
	<li>Kirkpatrick, BE and Rashkin, MD. Ancestry Testing and the Practice of Genetic Counseling. J Gen Counseling. 2016, Vol. 26, 1 (6-20).</li>
	<li>Hamburg, MA and Collins, FS. The Path to Personalized Medicine. N Engl J Med . 2010; , Vol. 363, 301-304.</li>
</ol>

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

Education

JoVE Lab Manual

Biologie

Genetics

Genetica

DNA-isolatie en restrictie-enzymanalyse

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 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

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Volg stap voor stap de procedures van DNA Isolation and Restriction Enzyme Analysis in deze 13 minuten durende studentprotocolvideo. Onderdeel van de Biology-collectie op JoVE Lab Manual.

Studentprotocol voor DNA Isolation and Restriction Enzyme Analysis in Biology - 33K+ weergaven | 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

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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 ...

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

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Biofuels: Producing Ethanol from Cellulosic Material

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
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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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