Recombinant DNA

an image showing the steps of cloning in bacterial cells.
An illustration depicting cloning using a plasmid containing LacZ (image attributed to “The Science Creative Quarterly” and Jen Philpot).

This unit will cover some basic recombinant DNA technologies, why they were developed, and how they are used today in many different scientific arenas. We will introduce the different types of restriction enzymes, their advantages and disadvantages, and how they are used to create a recombinant DNA molecule. We will review how to make a recombinant genomic DNA library and how to use this library to find a specific gene. We will then outline how to use a library to clone a gene by complementation of a mutant phenotype. We will also review how to make a recombinant cDNA library and how to use this library to find a specific gene. In addition, we will outline the differences between a genomic and a cDNA library and discuss how to use a cDNA library to clone a gene of interest. Finally, we will discuss methods to analyze genes identified through recombinant DNA technologies.

During this unit, you will learn the steps involved in a basic cloning strategy. You will determine which restriction enzyme to use to create a desired piece of recombinant DNA, given specific DNA sequences. You will also learn the function of DNA ligase, understand how vectors are used, and learn how to construct a recombinant genomic DNA library. You will then demonstrate how to use a genomic library to identify a gene of interest, and how to clone the gene of interest by complementation of a mutant phenotype. Next, you will learn how a recombinant cDNA library is constructed, and how it is used to identify a gene of interest by the protein made. You will move on to learning about the uses of agarose gel electrophoresis to analyze DNA molecules, and learn the basic mechanism of DNA sequencing by the dideoxy chain termination method. Finally, you will learn about the polymerase chain reaction (PCR) method of amplifying DNA, and when to use this technique.

By the end of this unit, you will understand some basic techniques of recombinant DNA technologies, and some of their applications. You will understand the differences between the techniques you have learned about, as well as their uses and their limitations. Your knowledge of recombinant DNA technologies will culminate in your understanding of the importance of GenBank^®, the vast database containing the DNA sequence of the entire genomes for many different organisms, and understanding why this is useful.

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Session Overview

This session will outline methods to analyze genes identified through recombinant DNA technologies.

Learning Objectives

To understand what an agarose gel is and how to use agarose gel electrophoresis to analyze DNA molecules.
To understand the basic mechanism of DNA sequencing by the dideoxy chain termination method.
To know that there is a vast database containing the DNA sequence of the entire genomes for many different organism, and understand why this is useful.
To understand the basic mechanism of the polymerase chain reaction (PCR) method of amplifying DNA and know when to use this technique.

Session Activities

Lecture Video

Watch the lecture video excerpt

Agarose Gel Electrophoresis, DNA Sequencing, PCR, Excerpt 1

Check Yourself

Question 1

Select the true statement or statements.

Agarose gel electrophoresis separates DNA molecules by size. check

When using Agarose gel electrophoresis, DNA molecules move toward the negative electrode. close

When using Agarose gel electrophoresis, large DNA molecules move faster through the gel. close

Question 2

When using chain termination sequencing, the nucleotides added as a chain terminator are…

missing the hydroxyl on both the 2’ and 3’carbon check

missing the hydroxyl on the 2’ carbon close

missing the hydroxyl on the 3’ carbon close

Session Activities

Lecture Video

Watch the lecture video excerpt

Agarose Gel Electrophoresis, DNA Sequencing, PCR, Excerpt 2

Check Yourself

Question 1

Which of the following are required for a successful PCR amplification? 1. One or more copies of the DNA to be amplified. 2. all of the normal DNA nucleotides. 3. some dideoxynucleotides 4. Two distinct primers 5. E. coli DNA polymerase 6. DNA polymerase from an organism that lives at high temperature

All of these are required close

a, b, d, f check

a, c, d, e close

a, c, d, f close

b, c, d, e close

Session Activities

Help Session Video

Watch the short video of Robert Dorkin explaining the Polymerase Chain Reaction (PCR) and DNA sequencing.

Polymerase Chain Reaction (PCR)

Practice Problems

Further Study

Useful Links

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Session Overview

This session will cover 1) what restriction enzymes are and how they cut DNA, 2) the different types of restriction enzymes and the advantages and disadvantages of using them, and 3) how restriction enzymes are used to create a recombinant DNA molecule.

Learning Objectives

To understand what a restriction endonuclease (restriction enzyme) is and how it functions.
To identify restriction enzyme(s) recognition sites given a section of DNA.
To understand the difference between enzymes that cut double-stranded DNA to produce a region of single-stranded DNA and those that do not, and the appropriate use of each type of enzyme.
To determine which restriction enzyme to use to create a desired piece of recombinant DNA.
To know the function of DNA ligase.
To understand what a vector is, what the minimum requirements are, and how it is used.

Session Activities

Lecture Video

Watch the lecture video excerpt

Basic Mechanisms of Cloning, Excerpt 1

Check Yourself

Question 1

Select the true statement or statements. 1. Restriction enzymes break a phosphodiester bond on only one of the two DNA strands. 2. Restriction enzymes break a phosphodiester bond on both of the DNA strands. 3. All restriction enzymes produce a region of single-stranded DNA. 4. Two different linear DNA molecules are cut with a single restriction enzyme such that only one end of each molecule is cut and these ends have regions of single stranded DNA. The two different DNA molecules will have compatible ends. 5. Two different linear DNA molecules are each cut with a different restriction enzyme such that only one end of each molecule is cut and these ends have regions of single stranded DNA. The two different DNA molecules will NOT have compatible ends.

a,c close

a,d close

b,d check

c,e close

Session Activities

Lecture Video

Watch the lecture video excerpt

Basic Mechanisms of Cloning, Excerpt 2

Check Yourself

Question 1

Which of the following features are essential in a vector that will be used to make many copies of the human gene X in bacterial cells? 1. A restriction enzyme site 2. A human origin of replication 3. A bacterial origin of replication 4. A bacterial selectable marker

a,b,c close

a,b,d close

a,c,d check

b,d close

c,d close

Session Activities

Lecture Video

Watch the lecture video excerpt

Basic Mechanisms of Cloning, Excerpt 3

Check Yourself

You want to:

Obtain the human insulin gene.
Cut chosen vector and ligate the human insulin gene into vector.
Use this ligation mixture to transform E. coli cells.
Transfer E. coli cells to growth media.
Select for E. coli cells that have obtained any vector.

Each of the steps listed above requires a specific DNA sequence found on the vector.

Question 1

In the steps above, where is the restriction enzyme recognition site first needed?

Step 1 close

Step 2 check

Step 3 close

Step 4 close

Step 5 close

Question 2

In the steps above, where is the ampicillin resistance gene first needed?

Step 1 close

Step 2 close

Step 3 close

Step 4 close

Step 5 check

Question 3

In the steps above, where is the bacterial origin of replication first needed?

Step 1 close

Step 2 close

Step 3 close

Step 4 check

Step 5 close

Session Activities

Practice Problems

Further Study

Useful Links

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Session Overview

This session will review how to make a recombinant cDNA library and how to use this library to find a specific gene. This session will outline the differences between a genomic and a cDNA library, and discuss how to use a cDNA library to clone a gene of interest.

Learning Objectives

To understand what a recombinant cDNA library is and how it is constructed.
To understand how to use a cDNA library to identify a gene of interest by the protein made.

Session Activities

Lecture Video

Watch the lecture video excerpt

cDNA Libraries and Expression Libraries

Check Yourself

Question 1

A recombinant vector in a yeast genomic library may contain more than one gene. This is not the case with the recombinant vectors in a yeast cDNA library because…

different restriction enzymes are used. close

every cDNA fragment cloned into each vector carries only one gene. check

the host cells cannot be transformed with a vector carrying more than one gene. close

the vectors used in making a cDNA library are different. close

For a cDNA library, double stranded DNA is made from mRNA. In eukaryotes, each mRNA molecule represents only one gene.

Question 2

Unlike a genomic DNA library, particular care must be taken to ensure that the cDNA library represents every gene in the genome. Why is this the case?

A cDNA library lacks introns. close

Host cells are more likely to reject vectors with cDNA as compared to genomic DNA. close

It is harder to clone cDNA fragments as compared to genomic fragments into the vectors. close

Some genes are transcribed only in certain cell types or at low levels. check

A cDNA library is made from mRNA. A gene that is not actively transcribed in the cells used as a source of mRNA will not produce mRNA, so that gene will not be represented in the library.

Question 3

Many cDNA libraries are used as expression libraries. The vector chosen for use in an expression library must have additional DNA sequence that is not required in the vector chosen for use in a genomic DNA library. What is this sequence?

a promoter specific for the host organism check

a second selectable marker close

additional restriction enzyme sites close

an origin of replication close

Session Activities

Help Session Video

Watch the short video of Robert Dorkin explaining transformation and protein expression.

Transformation and Protein Expression

Practice Problems

Further Study

Useful Links

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Session Overview

This session will review how to make a recombinant genomic DNA library and how to use this library to find a specific gene. This session will outline using a library to clone a gene by complementation of a mutant phenotype.

Learning Objectives

To understand what a recombinant genomic DNA library is and how it is constructed.
To identify how to use a genomic library to identify a gene of interest.
To understand how to clone the gene of interest by complementation of a mutant phenoptype.
To understand the limitations of a genomic library.

Session Activities

Lecture Video

Watch the lecture video excerpt

Constructing and Screening a Recombinant DNA Library

Check Yourself

Question 1

Put the following steps in the appropriate order for constructing a genomic library: 1. Ligate the vector and insert DNA together using DNA ligase. 2. Isolate genomic DNA from organism. 3. Transform a population of host cells (E. coli). 4. Cut both the genomic DNA and the chosen vector with the same restriction enzyme(s). 5. Plate the transformed cells onto selective media.

a-b-c-d-e close

b-a-d-c-e close

b-d-a-c-e check

c-e-a-b-d close

d-a-c-b-e close

Question 2

You plan to create an E. Coli genomic DNA library and clone a gene for arginine synthesis (the ARG1 gene) by complementation of a mutant phenotype. Which of the following statements is true? 1. You would isolate genomic DNA from a wild type cell. 2. You would isolate genomic DNA from an ARG1- mutant cell. 3. You would transform a population of wild type host cells. 4. You would transform a population of ARG1- mutant cells. 5. You would plate the transformed cells onto media with ampicillin. 6. You would plate the transformed cells onto media without arginine.

All of these statements are true close

a, d, e close

a, d, f check

b, c, e close

b, c, f close

Session Activities

Help Session Video

Watch the short video of Robert Dorkin explaining genomic libraries and cDNA libraries.

Genomic and cDNA Libraries

Practice Problems

Further Study

Useful Links

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Session Overview

This session will outline why recombinant DNA technologies were developed and how they are used today in many different scientific arenas.

Learning Objectives

To understand some of the applications of recombinant DNA technologies.
To have an overview of the steps involved in a basic cloning strategy.

Session Activities

Lecture Video

Watch the lecture video excerpt

Overview of Recombinant DNA, Excerpt 1

Check Yourself

Question 1

Arrange the following steps in the correct order 1. Screen the bacterial colonies 2. Transform DNA into host bacterial cells 3. Cut DNA of interest 4. Select bacterial host cells that have been transformed 5. Paste DNA of interest into vectors

a, c, b, d, e close

c, b, e, d, a close

c, e, b, d, a check

e, c, d, b, a close

Session Activities

Lecture Video

Watch the lecture video excerpt

Overview of Recombinant DNA, Excerpt 2

Further Study

Useful Links

History of Genentech

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Session Overview

For this exam, you will need to know restriction enzymes and how they are used. You will also need to know the steps for constructing a DNA library, and cloning a gene of interest. In addition, you will need to know the steps for amplification of a sequence of DNA. Lastly, you will need to know how to analyze a plasmid using gel electrophoresis.

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Exam and Solutions

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