Recombinant DNA Technology

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Questions and Answers

What is the key step for incorporating the desired DNA fragment into the vector?

  • Digestion
  • Ligation (correct)
  • Transformation
  • Dephosphorylation

What is the purpose of dephosphorylation in DNA cloning?

  • To enhance the transformation efficiency
  • To cleave the vector at specific sites
  • To prevent the vector from self-ligating (correct)
  • To activate the DNA fragment for ligation

What is the role of the host organism in DNA cloning?

  • To replicate the recombinant DNA molecule (correct)
  • To act as a source of DNA fragments
  • To facilitate dephosphorylation of DNA fragments
  • To provide the necessary restriction enzymes

What is the purpose of digestion in DNA cloning?

<p>To create complementary sticky ends in the vector and DNA fragment (C)</p> Signup and view all the answers

What is the role of transformation in DNA cloning?

<p>To introduce the recombinant DNA molecule into the host cell (C)</p> Signup and view all the answers

What is the effect of introducing recombinant DNA encoding a protein into a host organism?

<p>The recombinant protein may not be produced. (D)</p> Signup and view all the answers

What is the purpose of using ampicillin and tetracycline in the selection of transformed cells?

<p>To prevent the growth of non-transformed cells. (B)</p> Signup and view all the answers

Which of the following is NOT a requirement for the expression of foreign proteins in a host organism?

<p>The use of an antibiotic for cell selection. (C)</p> Signup and view all the answers

In a transformation experiment, cells that are transformed with a non-recombinant vector will:

<p>Grow in both ampicillin and tetracycline containing medium. (B)</p> Signup and view all the answers

What is the process called where a recombinant molecule is analyzed to find clones with desired DNA inserts and biological properties?

<p>Screening (B)</p> Signup and view all the answers

What is the purpose of replicating the recombinant molecule inside a host cell?

<p>To increase the number of copies of the recombinant molecule (D)</p> Signup and view all the answers

What is the key difference between non-transformed and transformed cells in the context of this experiment?

<p>Transformed cells can grow in the presence of antibiotics while non-transformed cells cannot. (A)</p> Signup and view all the answers

After replication, what is the next step in the process?

<p>Screening (B)</p> Signup and view all the answers

What is the purpose of selecting recombinant colonies?

<p>To eliminate colonies that do not contain the recombinant molecule (D)</p> Signup and view all the answers

Why is it important to express the recombinant DNA?

<p>To produce the desired protein or other product (A)</p> Signup and view all the answers

What is the term for the production of multiple copies of a recombinant molecule?

<p>Replication (D)</p> Signup and view all the answers

What is the purpose of analyzing the recombinant molecule?

<p>To confirm that the DNA insert is present in the recombinant molecule (B)</p> Signup and view all the answers

What is the relationship between the process of screening and the process of expression?

<p>Screening identifies colonies that express the recombinant DNA (D)</p> Signup and view all the answers

Which vector is most suitable for cloning and expressing genes in mammalian cells?

<p>SV40 (C)</p> Signup and view all the answers

What is the main characteristic of a shuttle vector?

<p>It can replicate in multiple host organisms (A)</p> Signup and view all the answers

Which of these vectors is a viral vector?

<p>Baculovirus (A), SV40 (C), λgt11 (D)</p> Signup and view all the answers

Which vector is commonly used for large-scale protein production?

<p>Baculovirus (B)</p> Signup and view all the answers

Which vector is used to produce recombinant proteins in E. coli?

<p>λgt11 (D)</p> Signup and view all the answers

Why is it important to use a vector with an origin of replication?

<p>To allow the vector to replicate independently of the host genome (B)</p> Signup and view all the answers

What is the purpose of using restriction enzymes in cloning?

<p>To create a single, specific cut in the DNA sequence (C)</p> Signup and view all the answers

What is the role of a promoter in a vector?

<p>To control the expression of cloned genes (C)</p> Signup and view all the answers

What is the main advantage of using a baculovirus as a vector for protein expression?

<p>It can produce high yields of proteins (A)</p> Signup and view all the answers

Which of these factors does NOT influence the choice of a vector for cloning?

<p>The DNA sequence of the insert (D)</p> Signup and view all the answers

What is the primary function of DNA sequencing?

<p>To determine the linear order of nucleotide bases in DNA (C)</p> Signup and view all the answers

What is the significance of the introduction of recombinant DNA technology in the development DNA sequencing?

<p>Recombinant DNA technology provided the means to isolate and amplify specific DNA fragments, making sequencing more efficient and accurate. (A)</p> Signup and view all the answers

How does recombinant DNA technology impact gene expression?

<p>All of the above (D)</p> Signup and view all the answers

What is the effect of a recombinant DNA fragment containing an active promoter being located next to a previously silent host cell gene?

<p>The host cell gene is activated and starts expressing its protein. (B)</p> Signup and view all the answers

How does insertional inactivation affect gene expression?

<p>It disrupts the function of a gene involved in regulating gene expression. (C)</p> Signup and view all the answers

When did the development of rapid and efficient methods for DNA sequencing first take place?

<p>Mid 1970s (C)</p> Signup and view all the answers

What is the fundamental principle underlying DNA sequencing?

<p>The determination of the linear order of nucleotide bases in DNA (A)</p> Signup and view all the answers

What is the significance of the linear order of nucleotide bases in DNA?

<p>It encodes the genetic information for an organism (D)</p> Signup and view all the answers

When was the first human hormone produced by recombinant DNA technology?

<p>1978 (D)</p> Signup and view all the answers

Recombinant DNA technology is used to create what type of DNA?

<p>Artificially created DNA (D)</p> Signup and view all the answers

What is a key property of plasmids that contributes to their use as vectors in recombinant DNA?

<p>All of the above. (D)</p> Signup and view all the answers

What are the two main components involved in constructing recombinant DNA?

<p>A foreign DNA fragment and a plasmid vector (C)</p> Signup and view all the answers

What happens to the gene in the plasmid when a foreign DNA fragment is inserted?

<p>The foreign DNA fragment disrupts the expression of the gene. (C)</p> Signup and view all the answers

What does the term "DNA cloning" refer to?

<p>Producing multiple identical copies of a specific DNA fragment. (A)</p> Signup and view all the answers

What role do restriction enzymes play in recombinant DNA technology?

<p>They cleave DNA at specific sequences, creating fragments suitable for recombination. (C)</p> Signup and view all the answers

What is the primary function of DNA ligase in the context of recombinant DNA technology?

<p>To join two DNA fragments together, creating a phosphodiester bond. (D)</p> Signup and view all the answers

What is meant by the term "chimeric DNA"?

<p>A DNA molecule containing sequences from multiple distinct sources. (B)</p> Signup and view all the answers

What is one of the main advantages of using recombinant DNA technology to produce insulin?

<p>All of the above. (D)</p> Signup and view all the answers

What is the characteristic property of EcoRI restriction enzyme?

<p>It recognizes a specific sequence of nucleotides and cuts the DNA at a specific site. (A)</p> Signup and view all the answers

What type of ends are produced by HaeIII restriction enzyme?

<p>Blunt ends, which have no overhangs. (A)</p> Signup and view all the answers

What is the key aspect of DNA ligase's function?

<p>It joins two compatible ends of DNA fragments, creating a phosphodiester bond. (A)</p> Signup and view all the answers

What was the first event that led to the creation of recombinant DNA?

<p>The use of DNA ligase to join two DNA fragments. (B)</p> Signup and view all the answers

What type of bond does DNA ligase form to join two pieces of DNA?

<p>Phosphodiester bond (D)</p> Signup and view all the answers

Which of these statements BEST describes the impact of the Asilomar Conference on recombinant DNA technology?

<p>It stimulated discussion and development of guidelines for responsible use of recombinant DNA technology. (B)</p> Signup and view all the answers

Flashcards

Cloning Vector

A DNA molecule used to carry foreign DNA into a host cell.

DNA Fragment

A specific piece of DNA that is to be cloned.

Ligation

The process of joining two DNA fragments together using ligase enzyme.

Transformation

The introduction of recombinant DNA into a host organism.

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Selection of Recombinant DNA

Identifying host organisms that successfully incorporated the recombinant DNA.

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Recombinant DNA

DNA that has been artificially formed by combining constituents from different organisms.

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Host cell

A cell that supports the replication of a recombinant molecule.

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Recombinant colony

A group of cells that have successfully taken up recombinant DNA.

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Screening for clones

The process of identifying cells that contain desired recombinant DNA.

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DNA insert

A segment of DNA introduced into a host organism's genome.

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Expression of recombinant DNA

The process by which the inserted DNA is transcribed and translated into proteins.

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Molecule analysis

The examination of recombinant molecules to verify their properties and functionality.

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Replication

The process by which recombinant DNA is copied within a host cell.

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Recombinant DNA Expression

The process where foreign DNA is expressed in living cells to produce proteins.

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Transformed Colonies

Colonies of bacteria that have successfully incorporated recombinant DNA and can grow in selective media.

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Selective Media

Growth medium containing antibiotics like ampicillin or tetracycline to identify transformed organisms.

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Expression Vectors

Specialized plasmids used to promote high levels of protein production from foreign DNA.

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Recombinant Protein Production

The process of creating proteins from introduced recombinant DNA within host organisms.

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Recombinant DNA Technology

A method of combining DNA from different sources to create new genetic sequences.

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Cohen & Boyer

Researchers who first produced a recombinant DNA molecule, awarded the Nobel Prize in Chemistry in 1980.

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DNA Cloning

The process of making identical copies of a specific DNA fragment.

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Plasmid

A small circular DNA molecule within bacteria that can replicate independently.

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Restriction Enzyme

An enzyme that cuts DNA at specific nucleotide sequences to facilitate cloning.

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Blunt Ends

DNA fragments with no overhangs, cut straight across by restriction enzymes.

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Cohesive Ends

DNA fragments with short overhangs, allowing them to stick together.

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Gene Cloning

Making multiple identical copies of a specific gene.

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DNA Ligase

An enzyme that joins two pieces of DNA together by forming phosphodiester bonds.

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Nobel Prize in Chemistry 1980

Award given to Cohen and Boyer for their work on recombinant DNA.

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Asilomar Conference

A 1975 meeting that established guidelines for recombinant DNA research.

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First Human Hormone from rDNA

In 1978, the first human hormone (insulin) produced using recombinant DNA technology.

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Antibiotic Resistance Genes

Genes often carried by plasmids that confer resistance to antibiotics.

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Chimeric DNA

DNA formed from genetic material of two different species.

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Genetic Recombination

The process of combining genetic material from multiple sources to create new DNA sequences.

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Molecular Cloning Vector

A tool used to insert foreign DNA into a host organism for replication.

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YEp24

A shuttle vector that can replicate in E. coli and Saccharomyces cerevisiae.

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Viral Vector

Vectors derived from viruses designed to infect host cells for gene expression.

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λgt11

An expression vector derived from bacteriophage λ, used for gene cloning in E. coli.

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SV40

A virus that infects mammalian cells, used as a vector for gene cloning.

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Baculovirus

A viral vector that infects insect cells for high protein expression.

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Cloning Steps

The protocol involves digesting DNA, using vectors, and inserting foreign genes.

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Foreign DNA

DNA that is introduced into a host organism for cloning or expression.

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Active Promoter

A region of DNA that initiates transcription of a gene.

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Insertional Inactivation

Disruption of a gene's expression by inserting foreign DNA.

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Silent Gene

A gene that is not expressed or is inactive in a certain condition.

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DNA Sequencing

The process of determining the order of nucleotides in DNA.

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Nucleotide Bases

The building blocks of DNA; adenine, thymine, cytosine, guanine.

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DNA Sequencing History

The development of methods to sequence DNA began in the 1970s.

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Study Notes

Recombinant DNA Technology

  • Recombinant DNA is artificially created DNA, combining two or more sequences not normally found together
  • Recombinant DNA technology joins DNA molecules from different species, creating new genetic combinations for science, medicine, agriculture, and industry
  • Recombinant organisms have a different combination of alleles compared to their parents

Recombinant DNA

  • A recombinant organism is an organism whose DNA consists of a different combination of alleles compared to either of its parents
  • A recombinant virus is a virus formed by combining genetic material
  • Recombinant DNA is an artificial form of DNA

Recombinant DNA Technology

  • Scientists use restriction enzymes to cut DNA from different species at specific sites, and then fuse the cut strands together
  • The first recombinant DNA molecules were produced in 1972
  • The first publications on recombinant DNA technology were in 1972 and 1973

Important Events in Recombinant DNA Technology

  • 1972: DNA ligase joined two fragments, creating the first recombinant DNA molecules
  • 1973: Concerns arose about the potential hazards of recombinant DNA technology
  • 1974: A global moratorium on some recombinant experiments was implemented
  • 1975: Guidelines for recombinant DNA technology were developed at the Asilomar Conference
  • 1978: The first human hormone (insulin) was produced using recombinant DNA technology
  • 1980: The first patent for recombinant DNA technology was granted to Stanley N. Cohen and Herbert Boyer

Bacterial DNA

  • Most bacteria have one circular chromosome ranging from 1,000 to 8,000 kilobase pairs
  • Plasmids are extra-chromosomal genetic elements, also circular DNA molecules
  • A bacterium's complete genome includes the chromosome and plasmids

Plasmids

  • Plasmids replicate autonomously within a host
  • They often carry genes for resistance to antibiotics (e.g., tetracycline, ampicillin, kanamycin)
  • These marker genes are used to identify cells containing the plasmids

Recombinant DNA Technology

  • Recombinant DNA molecules are formed by combining genetic material from multiple sources using laboratory methods of genetic recombination (like molecular cloning)
  • The chemical structure of DNA is the same across all organisms; the difference lies in the nucleotide sequences

Gene Cloning

  • A clone is a collection of identical molecules or cells
  • Cloning a gene means creating many copies of it, often by replication in bacteria
  • Recombinant DNA technology enables gene manipulation

Restriction Enzymes

  • Restriction enzymes (endonucleases) cut DNA at specific nucleotide sequences
  • They produce blunt or sticky ends (cohesive ends)
  • Blunt ends have no overhangs, sticky ends have overhangs

DNA Ligase

  • DNA ligase joins two DNA pieces, catalyzing phosphodiester bond formation
  • It works with both blunt and sticky ends, joining double-stranded DNA

Cloning Vectors

  • Cloning vectors are DNA molecules that replicate inside living cells (often derived from plasmids or viruses)
  • They contain genetic signals for replication, elements for inserting foreign DNA, and elements to identify cells with recombinant DNA

Standard Cloning Protocols

  • Choosing a host organism and cloning vector
  • Preparing vector DNA
  • Preparing the DNA to be cloned (DNA insert)
  • Creating recombinant DNA
  • Introducing recombinant DNA into host organisms
  • Selecting organisms with recombinant DNA
  • Screening clones for desired DNA inserts and biological properties

Selection of Recombinant Colonies

  • Colonies are selected based on antibiotic resistance associated with antibiotic-containing media

Expression of Recombinant DNA

  • Recombinant DNA constructs are able to be expressed in living cells, resulting in recombinant proteins
  • Recombinant protein production requires specialized expression vectors.

Polymerase Chain Reaction (PCR)

  • A technique that amplifies DNA from very little starting material
  • PCR is a critical tool in recombinant DNA technology allowing the amplification of DNA from minute or degraded samples

Insertional Inactivation

  • Insertional inactivation occurs when recombinant DNA disrupts a gene, rendering it nonfunctional
  • This disruption can result from insertion into a gene's coding sequence

DNA Sequencing

  • DNA sequencing determines the order of nucleotide bases in a DNA fragment
  • The Sanger method (chain termination method) is widely used, and has been largely automated for faster, more efficient analysis.

Applications of Recombinant DNA

  • Forensics, medicine, agriculture, industry

Recombinant Chymosin

  • An enzyme used in cheese production
  • Genetically engineered chymosin is now commonly used to make cheese in industry today

Zebra Fish

  • Zebra fish have been genetically modified to detect pollution

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Introduction to Recombinant DNA Technology PDF

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