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By the late 1970s, techniques for easily sequencing ______, synthesizing oligonucleotides, and expressing eukaryotic genes in prokaryotes had also been developed.
By the late 1970s, techniques for easily sequencing ______, synthesizing oligonucleotides, and expressing eukaryotic genes in prokaryotes had also been developed.
DNA
Between 1983 and 1985, Kary Mullis developed a new technique to synthesize large quantities of a DNA fragment without ______ it.
Between 1983 and 1985, Kary Mullis developed a new technique to synthesize large quantities of a DNA fragment without ______ it.
cloning
This is the polymerase chain reaction or ______ technique.
This is the polymerase chain reaction or ______ technique.
PCR
The first step in the PCR technique is to ______ the DNA at 94°C for 15 seconds.
The first step in the PCR technique is to ______ the DNA at 94°C for 15 seconds.
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The third step in the PCR technique is ______ at 68°C for 60 seconds.
The third step in the PCR technique is ______ at 68°C for 60 seconds.
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The easiest vectors to work with are ______.
The easiest vectors to work with are ______.
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RDNA phages and other viruses are more conveniently stored for long ______.
RDNA phages and other viruses are more conveniently stored for long ______.
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Larger pieces of DNA can be cloned with cosmids and artificial ______.
Larger pieces of DNA can be cloned with cosmids and artificial ______.
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______ is synthesized for the treatment of diabetes.
______ is synthesized for the treatment of diabetes.
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Erythropoeitin is used in the treatment of ______.
Erythropoeitin is used in the treatment of ______.
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Diagnosis of ______ disease can be done using genetic engineering.
Diagnosis of ______ disease can be done using genetic engineering.
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Howard Temin and David Baltimore discovered the enzyme called ______.
Howard Temin and David Baltimore discovered the enzyme called ______.
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The restriction enzyme ______, isolated from E. coli, cleaves the DNA between G and A in the base sequence GAATTC.
The restriction enzyme ______, isolated from E. coli, cleaves the DNA between G and A in the base sequence GAATTC.
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The EcoRI enzyme cleaves DNA leaving two ______ ends.
The EcoRI enzyme cleaves DNA leaving two ______ ends.
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Reverse transcriptase can be used to construct a DNA copy called ______.
Reverse transcriptase can be used to construct a DNA copy called ______.
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The restriction enzyme EcoRI cleaves the DNA between G and ______ in the base sequence GAATTC.
The restriction enzyme EcoRI cleaves the DNA between G and ______ in the base sequence GAATTC.
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The mRNA strand ends with a ______ tail.
The mRNA strand ends with a ______ tail.
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Once fragments have been isolated, they are ligated with an appropriate ______, such as a plasmid.
Once fragments have been isolated, they are ligated with an appropriate ______, such as a plasmid.
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The first step is to cut the plasmid with the restriction enzyme ______.
The first step is to cut the plasmid with the restriction enzyme ______.
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The plasmid and donor DNA are joined together by the enzyme ______, forming a recombinant DNA molecule.
The plasmid and donor DNA are joined together by the enzyme ______, forming a recombinant DNA molecule.
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The ______ DNA is also cut with the restriction enzyme EcoRI, creating complementary sticky ends.
The ______ DNA is also cut with the restriction enzyme EcoRI, creating complementary sticky ends.
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The recombinant DNA molecule is introduced into a ______ cell, which is then grown in a medium containing tetracycline.
The recombinant DNA molecule is introduced into a ______ cell, which is then grown in a medium containing tetracycline.
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One of the easiest approaches is to cut the plasmid and donor DNA with the same ______ enzyme.
One of the easiest approaches is to cut the plasmid and donor DNA with the same ______ enzyme.
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Cells that have integrated the recombinant DNA molecule will be resistant to ______ and will form colonies on the petri dish.
Cells that have integrated the recombinant DNA molecule will be resistant to ______ and will form colonies on the petri dish.
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This molecule can be transformed into a bacterial cell, where it can be transcribed and ______.
This molecule can be transformed into a bacterial cell, where it can be transcribed and ______.
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The next milestone was the successful generation of recombinant DNA ______.
The next milestone was the successful generation of recombinant DNA ______.
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They allowed the sticky ends of fragments to anneal then covalently joined the fragments with the enzyme DNA ______.
They allowed the sticky ends of fragments to anneal then covalently joined the fragments with the enzyme DNA ______.
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Within a year, plasmid ______, or carriers of foreign DNA fragments during gene cloning, had been developed and combined with foreign DNA.
Within a year, plasmid ______, or carriers of foreign DNA fragments during gene cloning, had been developed and combined with foreign DNA.
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DNA cut by a restriction ______.
DNA cut by a restriction ______.
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Discovery of Southern blotting technique for detecting specific DNA fragments so that a particular gene could be ______ from a complex DNA mixture.
Discovery of Southern blotting technique for detecting specific DNA fragments so that a particular gene could be ______ from a complex DNA mixture.
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The Southern blotting technique depends on the specificity of base ______ in nucleic acids.
The Southern blotting technique depends on the specificity of base ______ in nucleic acids.
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They normally protect the host cell by destroying phage DNA after its ______.
They normally protect the host cell by destroying phage DNA after its ______.
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Cells protect their own DNA from restriction enzymes by ______ nucleotides in the sites that these enzymes recognize.
Cells protect their own DNA from restriction enzymes by ______ nucleotides in the sites that these enzymes recognize.
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Incoming foreign DNA is not methylated at the same sites and often is ______ by host restriction enzymes.
Incoming foreign DNA is not methylated at the same sites and often is ______ by host restriction enzymes.
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There are ______ general types of restriction enzymes.
There are ______ general types of restriction enzymes.
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Types I and III cleave DNA away from ______ sites.
Types I and III cleave DNA away from ______ sites.
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Type II restriction endonucleases cleave DNA at specific ______ sites.
Type II restriction endonucleases cleave DNA at specific ______ sites.
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The type II enzymes can be used to prepare DNA fragments containing specific ______ or portions of genes.
The type II enzymes can be used to prepare DNA fragments containing specific ______ or portions of genes.
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EcoRI is a restriction enzyme derived from ______ coli that recognizes the sequence 5’-G-A-A-T-T-C-3’.
EcoRI is a restriction enzyme derived from ______ coli that recognizes the sequence 5’-G-A-A-T-T-C-3’.
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One can extract all the DNA from an organism, cleave the DNA into ______, isolate the fragment of interest for cloning.
One can extract all the DNA from an organism, cleave the DNA into ______, isolate the fragment of interest for cloning.
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All of the fragments can be cloned by means of a suitable ______, and each clone tested for the desired gene.
All of the fragments can be cloned by means of a suitable ______, and each clone tested for the desired gene.
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A DNA fragment can directly be ______.
A DNA fragment can directly be ______.
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Agarose or polyacrylamide gels are used to separate DNA ______.
Agarose or polyacrylamide gels are used to separate DNA ______.
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The deliberate modification of an organism's genetic information by directly changing its nucleic acid genome is called ______.
The deliberate modification of an organism's genetic information by directly changing its nucleic acid genome is called ______.
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DNA molecules are placed in an electrical ______ and allowed to migrate toward the positive and negative poles.
DNA molecules are placed in an electrical ______ and allowed to migrate toward the positive and negative poles.
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By inserting recombinant DNA molecules into an organism, these molecules can be ______.
By inserting recombinant DNA molecules into an organism, these molecules can be ______.
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The molecules separate because they move at different rates due to their differences in ______ and size.
The molecules separate because they move at different rates due to their differences in ______ and size.
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Recombinant DNA technology opens up totally new areas of ______ and applied biology.
Recombinant DNA technology opens up totally new areas of ______ and applied biology.
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Negatively charged DNA fragments move toward the positive electrode or ______.
Negatively charged DNA fragments move toward the positive electrode or ______.
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One of the first breakthroughs leading to recombinant DNA technology was the discovery of microbial enzymes called ______.
One of the first breakthroughs leading to recombinant DNA technology was the discovery of microbial enzymes called ______.
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Each fragment’s migration rate is inversely proportional to the log of its ______ weight.
Each fragment’s migration rate is inversely proportional to the log of its ______ weight.
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These enzymes recognize and cleave specific sequences about ______ base pairs long.
These enzymes recognize and cleave specific sequences about ______ base pairs long.
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Recombinant DNA is DNA with a new sequence formed by joining fragments from two or more different ______.
Recombinant DNA is DNA with a new sequence formed by joining fragments from two or more different ______.
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Study Notes
Recombinant DNA Technology
- Recombinant DNA technology allows for the direct manipulation of DNA, enabling the deliberate modification of an organism's genetic information.
- This technology is accomplished by a collection of methods known as recombinant DNA technology.
Historical Perspectives
- The discovery of restriction enzymes in the late 1960s by Werner Arber and Hamilton Smith was a crucial breakthrough leading to recombinant DNA technology.
- These enzymes recognize and cleave specific sequences about 4 to 8 base pairs long and are known as restriction enzymes or restriction endonucleases.
- There are three general types of restriction enzymes:
- Type I and III cleave DNA away from recognition sites.
- Type II restrictions endonucleases cleave DNA at specific recognition sites.
- Type II enzymes can be used to prepare DNA fragments containing specific genes or portions of genes.
Polymerase Chain Reaction (PCR)
- Kary Mullis developed the PCR technique between 1983 and 1985.
- PCR is a technique to synthesize large quantities of a DNA fragment without cloning it.
- The steps involved in PCR are:
- Denaturation at 94°C for 15 seconds.
- Annealing.
- Extension at 68°C for 60 seconds.
Cloning Vectors
- There are four major types of vectors:
- Plasmids.
- Bacteriophages and other viruses.
- Cosmids.
- Artificial chromosomes.
- Plasmids are the easiest to work with.
- rDNA phages and other viruses are more conveniently stored for long periods.
- Larger pieces of DNA can be cloned with cosmids and artificial chromosomes.
Medical Applications
- Recombinant DNA technology has various medical applications, including:
- Synthesis of medically important molecules, such as:
- α₁-antitrypsin for treatment of emphysema.
- Interferons for antiviral, antitumor, and anti-inflammatory agents.
- Blood-clotting factor VIII for treatment of hemophilia.
- Diagnosis of infectious diseases.
- Synthesis of medically important molecules, such as:
Isolating and Cloning Fragments
- Once fragments have been isolated, they are ligated with an appropriate vector to form a recombinant molecule.
- This can then be introduced into the host.
- One of the easiest and most popular approaches is to cut the plasmid and donor DNA with the same restriction enzyme so that identical sticky ends are formed.
- The steps involved in isolating and cloning fragments are:
- Cutting the plasmid with a restriction enzyme, such as EcoRI.
- Cutting the donor DNA with the same restriction enzyme.
- Ligation of the sticky ends.
- Transformation of the recombinant DNA molecule into a bacterial cell.
- Selection of bacteria that have integrated the recombinant DNA molecule into their genome.
Electrophoresis
- Agarose or polyacrylamide gels are used to separate DNA fragments.
- DNA molecules are placed in an electrical field and allowed to migrate toward the positive and negative poles.
- The molecules separate because they move at different rates due to their differences in charge and size.
Preparation of Recombinant DNA
- There are three ways to obtain adequate quantities of a DNA fragment:
- Extracting all the DNA from an organism, cleaving it into fragments, and isolating the fragment of interest for cloning.
- Cloning all the fragments and testing each clone for the desired gene.
- Directly synthesizing a DNA fragment.
Restriction Enzyme
- The restriction enzyme EcoRI, isolated from E. coli, cleaves the DNA between G and A in the base sequence GAATTC.
- Leaving two sticky ends or cohesive ends.
Making cDNA
- The enzyme reverse transcriptase can be used to construct a DNA copy, called complementary DNA (cDNA), of any RNA.
- The process of making cDNA involves:
- Reverse transcription of mRNA to form a cDNA with a hairpin loop.
- Removing the RNA using RNase H or alkali.
- Synthesizing the second strand using DNA polymerase I and free nucleotides.
- Removing the hairpin loop using S1 nuclease.
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Description
Learn about the direct manipulation of DNA and the deliberate modification of an organism's genetic information through recombinant DNA technology. Discover its history and key breakthroughs.
