Name: steps in dna extraction
Uploaded: 2026-08-26T08:20:38.799Z
Duration: 1 min 58 s
Channel: JoVE

A extra&#xE7;&#xE3;o de DNA &#xE9; a remo&#xE7;&#xE3;o e purifica&#xE7;&#xE3;o do DNA das c&#xE9;lulas. Primeiro, as c&#xE9;lulas s&#xE3;o lisadas, abertas, geralmente por meio de uma combina&#xE7;&#xE3;o de perturba&#xE7;&#xE3;o f&#xED;sica e tratamento com produtos qu&#xED;micos, como detergentes, que dissolvem a c&#xE9;lula e as membranas nucleares. O conte&#xFA;do pode flutuar livremente na solu&#xE7;&#xE3;o circundante.Em seguida, o DNA deve ser separado das outras mol&#xE9;culas presentes. Solventes org&#xE2;nicos, tal como o fenol e o clorof&#xF3;rmio, s&#xE3;o comumente usados para separar &#xE1;cidos nucleicos de prote&#xED;nas com base em sua solubilidade diferente. Al&#xE9;m disso, proteinase e enzimas RNase podem ser usadas para degradar prote&#xED;nas e RNA, deixando o DNA intacto.O sal &#xE9; geralmente adicionado &#xE0; amostra, que estabiliza os grupos de fosfato negativamente carregados na espinha dorsal do DNA, e depois a adi&#xE7;&#xE3;o de &#xE1;lcool gelado, precipita o DNA da solu&#xE7;&#xE3;o. O precipitado branco &#xE9; coletado girando-o em uma centr&#xED;fuga, onde se instala para a parte inferior do tubo. Depois de lavar e suspender em uma solu&#xE7;&#xE3;o tamp&#xE3;o, o DNA extra&#xED;do pode finalmente ser usado em aplica&#xE7;&#xF5;es de pesquisa ou biotecnologia.

isolamento do dna

DNA Isolation

O DNA das c&#xE9;lulas &#xE9; necess&#xE1;rio para muitas aplica&#xE7;&#xF5;es de biotecnologia e investiga&#xE7;&#xE3;o, como a clonagem molecular. Para remover e purificar o DNA das c&#xE9;lulas, os investigadores usam v&#xE1;rios m&#xE9;todos de extra&#xE7;&#xE3;o de DNA. Embora as especificidades de diferentes protocolos possam variar, alguns conceitos gerais est&#xE3;o por tr&#xE1;s do processo de extra&#xE7;&#xE3;o de DNA.
 
 <h4>O Processo</h4>
 
 Primeiro, as c&#xE9;lulas precisam ser lisadas&#x2014;abertas&#x2014;para libertar o DNA em solu&#xE7;&#xE3;o. As c&#xE9;lulas podem ser fisicamente lisadas usando equipamentos como um homogeneizador, sonicador ou beadbeater, que moem ou aplicam for&#xE7;a para abrir as c&#xE9;lulas. Muitas vezes, subst&#xE2;ncias como detergentes s&#xE3;o adicionadas durante a lise para romper quimicamente as membranas celulares constitu&#xED;das por lip&#xED;dios&#x2014;ajudando a libertar o DNA do n&#xFA;cleo e da c&#xE9;lula. A utiliza&#xE7;&#xE3;o de uma centr&#xED;fuga sedimenta os detritos celulares no fundo, e o lisado&#x2014;contendo materiais celulares&#x2014;&#xE9; recolhido para processamento posterior.
 
 O DNA deve ent&#xE3;o ser separado de outras mol&#xE9;culas celulares, como o RNA e prote&#xED;nas. Portanto, enzimas como RNase e Proteinase K s&#xE3;o frequentemente adicionadas durante ou ap&#xF3;s a lise para degradar o RNA e as prote&#xED;nas, respectivamente. Al&#xE9;m disso, solventes org&#xE2;nicos como fenol e clorof&#xF3;rmio s&#xE3;o comumente usados para separar o DNA da prote&#xED;na. Tipicamente, a amostra &#xE9; vortexada com fenol-clorof&#xF3;rmio e, em seguida, centrifugada para separar as fases aquosas e org&#xE2;nicas no tubo. A fase aquosa no topo contendo DNA pode ent&#xE3;o ser pipetada, deixando para tr&#xE1;s a prote&#xED;na na fase org&#xE2;nica.
 
 Sais s&#xE3;o frequentemente adicionados durante a extra&#xE7;&#xE3;o de DNA para estabilizar o DNA e ajudar a precipit&#xE1;-lo da solu&#xE7;&#xE3;o. Um m&#xE9;todo padr&#xE3;o de precipita&#xE7;&#xE3;o de DNA &#xE9; adicionar &#xE1;lcool gelado, como etanol ou isopropanol, &#xE0; amostra. Isso faz com que o DNA forme um precipitado branco e nublado dentro do l&#xED;quido no tubo.
 
 A amostra &#xE9; ent&#xE3;o centrifugada, fazendo com que o DNA precipitado se junte na parte inferior do tubo em um pellet. O pellet &#xE9; lavado por remo&#xE7;&#xE3;o do l&#xED;quido, lavagem em &#xE1;lcool e nova centrifuga&#xE7;&#xE3;o. Ap&#xF3;s a lavagem final, o pellet &#xE9; ressuspendido em uma solu&#xE7;&#xE3;o tamp&#xE3;o&#x2014;criando uma solu&#xE7;&#xE3;o aquosa de DNA purificado que est&#xE1; pronto para ser estudado ou usado em biotecnologia.

Isolamento do DNA

DNA from cells is required for many biotechnology and research applications, such as molecular cloning. To remove and purify DNA from cells, researchers ...

https://cloudfront.jove.com/files/ftp_upload/10814/10814_article_thumb_10814.jpg

Education

JoVE Core

Biology

Biologia

Genetics

Genética

Biotechnology

Biotecnologia

KEY TERMS AND CONCEPTS

TERMOS-CHAVE E CONCEITOS

steps in dna extraction

DNA extraction is the process of removing and purifying DNA from cells. It is used in many biotechnology and research applications, including molecular cloning. The main goal is to free DNA from the cell and prepare it for later study or use.

The first step is cell lysis, which means breaking cells open. Researchers may use a homogenizer, sonicator, or bead beater to physically lyse the cells. They also often add detergent to chemically disrupt the lipid-based cell membranes. This helps release DNA from the nucleus and the rest of the cell.

After lysis, the sample is spun in a centrifuge. This causes cell debris to settle at the bottom, while the liquid lysate is kept for the next steps. The DNA then has to be separated from other cellular molecules, such as RNA and proteins. Enzymes like RNase and Proteinase K are often added to break down RNA and proteins, and phenol-chloroform can be used to separate DNA from protein.

During the phenol-chloroform step, the sample is vortexed and centrifuged again. The tube separates into an aqueous phase and an organic phase. The DNA stays in the aqueous phase at the top, and this layer can be pipetted off. Salts are often added to help stabilize the DNA and support precipitation.

To collect the DNA, ice-cold alcohol such as ethanol or isopropanol is added. The DNA then forms a white, cloudy precipitate in the tube. Another centrifuge spin pulls this DNA into a pellet at the bottom. The pellet is washed with alcohol, spun again, and then re-suspended in buffer to make a purified DNA solution ready for biotechnology work.

Steps in DNA Extraction

what is genetic engineering?

How Scientists Edit DNA for New Traits

Genetic engineering changes an organism’s DNA to add useful traits. Scientists use it in bacteria, plants, animals, and even viruses for research, medicine, agriculture, and industry. It can bring major benefits, but it also raises ethical concerns about humans and the food supply. This work is possible because DNA has the same basic structure and genetic code across life. That shared code lets scientists modify DNA in several ways. One method is CRISPR/Cas9, often called molecular scissors,...

dna isolation

DNA extraction is the process of removing and purifying DNA from cells. It is used in many biotechnology and research applications, including molecular cloning. The main goal is to free DNA from the cell and prepare it for later study or use. The first step is cell lysis, which means breaking cells open. Researchers may use a homogenizer, sonicator, or bead beater to physically lyse the cells. They also often add detergent to chemically disrupt the lipid-based cell membranes. This helps...

recombinant dna technology

Recombinant DNA in Cloning and GMOs

Recombinant DNA is made by joining DNA from different sources in the lab. Scientists often use it to clone genes and study them in cells that are easy to handle, such as bacteria. Organisms that carry recombinant DNA are called genetically modified organisms, or GMOs. This technology gives scientists new tools for research, medicine, and agriculture. To build recombinant DNA, researchers place a gene of interest into a vector, which is a DNA carrier that moves foreign DNA into a host cell. The...

PCR Cycle for DNA Amplification

PCR is a laboratory method for making many copies of a DNA segment. It can turn a small amount of DNA into millions or even billions of copies in just a few hours. The process uses a heat-resistant DNA polymerase inside a machine called a thermocycler. PCR was developed by Kary Mullis in 1983, and he received the 1993 Nobel Prize in Chemistry for it. The method is fast, relatively inexpensive, and precise. It has become useful for molecular cloning, gene mutagenesis, pathogen detection, gene...

complementary dna

From mRNA to cDNA for Gene Studies

Complementary DNA, or cDNA, is made from messenger RNA (mRNA) so scientists can study which genes are active in cells and tissues. Only genes that are transcribed into mRNA are expressed. By extracting mRNA from cells, researchers can compare gene expression across samples. cDNA is produced by reverse transcription. This process copies mRNA into DNA, which is useful because mRNA does not contain introns, or non-coding regions, and other regulatory sequences. As a result, cDNA can help...

antibiotic selection

Antibiotic Resistance in Cloning

Antibiotic resistance genes help scientists find bacteria that took up a plasmid with a gene of interest. In genetic engineering, this resistance is used as a marker, not just as a medical concern. The same process also connects to the growing problem of resistant bacteria in the real world. Antibiotics, such as penicillin, stop bacterial growth or kill bacteria. When bacteria naturally have, or are engineered to carry, an antibiotic resistance gene, the antibiotic no longer works on them.

transgenic organisms

Creating Transgenic Plants and Animals

Transgenic organisms are genetically engineered to carry a transgene, which is a gene from a different species, in their genome. The inserted gene may be a new gene for that organism or a different version of one it already has. Scientists usually make transgenes with recombinant DNA and DNA cloning methods. Researchers use transgenic bacteria, plants, and animals to answer biological questions and to build useful tools. To make a transgenic organism, they first choose a transgenesis method,...

in-vitro mutagenesis (gene knockouts)

Knockout Mice and Gene Replacement

Knockout mice help researchers study gene function by showing what happens when a gene is turned off. These engineered animals are also useful for modeling human diseases such as cancer, Parkinson’s disease, and diabetes. A specific gene can be targeted by using a targeting vector, which is a piece of engineered DNA that replaces the normal gene and inactivates it. The vector has matching DNA sequences on both ends. These homologous sequences let the vector swap in for the target gene through...

crispr

CRISPR-Cas9 Gene Editing in Action

CRISPR-Cas9 is a genome editing tool that lets scientists change DNA at a specific location. It can add, remove, or rearrange genetic material. Researchers study it because it may help treat genetic disorders such as hemophilia and sickle cell anemia. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and Cas9 is a CRISPR-associated protein 9. The basic system includes a Cas9 endonuclease, which is an enzyme that cuts DNA, and a small RNA that guides Cas9 to the...

gene therapy

Gene Delivery for Serious Diseases

Gene therapy is a technique used to treat serious disease by adding a gene to a person’s cells. The added gene may be a healthy copy of a mutated gene. It may also be a different gene that inactivates or compensates for the disease-causing gene. One example is severe combined immunodeficiency, or SCID, caused by a mutation in the gene for adenosine deaminase. In that case, a working copy of the gene can be inserted so the patient’s cells make the enzyme again. This can cure the disease in some...

embryonic stem cells

How Embryonic Stem Cells Become Specialized

Embryonic stem cells are pluripotent cells that have not yet specialized. Pluripotent means they can give rise to many different cell types in the body. Because of this, they are useful in science and medicine for making cells needed in research or for replacing cells lost to damage or disease. These cells come from the inner cell mass of a blastocyst, which is an early embryo about 3 to 5 days after fertilization in humans. At this stage, the embryo has not yet implanted in the uterus. Human...

adult stem cells

Adult Stem Cells in the Small Intestine

Adult stem cells help maintain tissues by replacing old or damaged cells. These undifferentiated cells divide to make more stem cells or progenitor cells, which then become mature, specialized cells. Small groups of adult stem cells are found in many tissues, including the bone marrow, brain, skin, and gut. The small intestine shows this process clearly. Its lining, called the epithelium, is renewed continuously by adult stem cells. It is also the fastest replaced tissue in the human body,...

induced pluripotent stem cells

Reprogramming Mature Cells into Stem Cells

Reprogramming mature cells into stem cells lets scientists turn specialized cells back into an unspecialized state. These cells are called induced pluripotent stem cells, or iPSCs. Like embryonic stem cells, iPSCs are pluripotent, which means they can form all cell types. To make iPSCs, researchers start with mature cells such as skin fibroblasts or blood cells. The cells are grown in culture, and genes for several transcription factors are delivered with a viral vector. The cells then make...

reproductive cloning

Cloning Animals with SCNT

Reproductive cloning can produce a genetically identical copy of an entire organism. In adult animals, this is usually done with somatic cell nuclear transfer, or SCNT. Another way clones can form is by splitting an early embryo, which is similar to how identical twins arise naturally. In SCNT, an egg cell is taken from an animal and its nucleus is removed. This creates an enucleated egg. A somatic cell, which is any body cell that is not a sex cell, is then taken from the animal to be cloned.

genomics

Genomics and DNA Sequencing

Genomics studies the full set of genetic material in an organism. In humans, that genome includes the DNA carried in 23 pairs of chromosomes in the nucleus, along with mitochondrial DNA. Genomics looks at both coding DNA and non-coding DNA, which helps scientists study how living things are built and how they differ. This field is useful in many areas of biology and medicine. Scientists use genomics to build phylogenetic trees, improve crop productivity and sustainability, help with criminal...

key terms and concepts

Learn the main steps in DNA extraction, including cell lysis, centrifugation, protein removal, and DNA precipitation for purified samples.

Steps in DNA Extraction | JoVE K12

JoVE Core series brings biology to life through over 300 concise and easy-to-understand animated video lessons that explain key concepts in biology, plus more than 150 scientist-in-action videos that show actual research experiments conducted in today’s laboratories.

biology

A coleção JoVE Core dá vida à biologia através de mais de 300 vídeos de animação concisos e fáceis de entender que explicam conceitos-chave de biologia, além de mais de 150 vídeos de Cientistas em ação que mostram experimentos reais em pesquisa realizados nos laboratórios atualmente.

Steps in DNA Extraction

Description

Transcript

Tags