Gene Cloning
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Who was Dolly?
The first successfully cloned organism that became widely reported.
When did the cloning of Dolly occur?
During the 1990s.
What was the reaction of the scientific community to Dolly’s cloning?
The scientific community was in uproar because of the successful cloning report.
What effect did Dolly’s cloning have on research?
Large amounts of funding were invested into cloning research.
What did researchers want to determine after Dolly’s cloning?
How cloning works.
What is cloning?
Producing identical copies.
In what field did the lecture emphasize the importance of cloning?
The medical field.
What can cloning produce?
- Specific parts 2. Organisms
What is the purpose of cloning?
To specify important features of an organism or organ.
What can happen once a desirable feature is identified?
Many copies of that product or organism can be produced through cloning.
What type of cloning holds great promise according to the lecture?
Gene cloning.
What are the applications of gene cloning?
- Identification of diseases 2. Disease control 3. Disease management 4. Addressing the occurrence of diseases
What is a clone?
A collection of molecules or cells identical to the original molecule or cell.
What does it mean to clone a gene?
To make many copies of a gene.
How can a gene be cloned?
By replicating it in a culture of bacteria.
What is a wild-type cloned gene?
A normal copy of a gene.
What is a mutant cloned gene?
An altered version of a gene.
What technology makes manipulation of genes possible?
Recombinant DNA technology.
Why do scientists prepare gene-sized pieces of DNA in identical copies?
To work directly with specific genes.
What is the process of preparing identical copies of DNA called?
DNA cloning.
What does a clone refer to in molecular biology?
A collection of molecules or cells identical to the original molecule or cell.
What is the first step in gene cloning according to the lecture?
Start with a gene fragment from the gene map.
What happens to the gene fragment during cloning?
It is replicated to produce multiple copies.
What happens to the cloned gene after replication?
It is cultured and introduced into a vector.
What can the cloned gene function as?
The wild-type gene.
What example of gene function was given?
Gene A converts a substrate into a product.
Why are multiple copies of Gene A needed?
To produce large amounts of the corresponding product.
Why is replicated DNA inserted into a vector?
To study gene expression and gene function.
How can scientists study gene function using cloning?
By cloning the native copy and introducing mutations.
What is observed after mutations are introduced into the cloned gene?
The effects of alterations on gene function.
What field arose from cloning and manipulation of genes?
Recombinant DNA technology (RDT).
What is recombinant DNA technology (RDT)?
The umbrella field for manipulating genes.
What does RDT allow scientists to do?
Work directly with specific genes using cloned fragments.
What are the steps in the cloning process?
- Prepare a gene fragment 2. Insert the fragment into a vector 3. Replicate the fragment to produce multiple copies 4. Study gene expression and function through the cloned gene
What are the basic requirements for cloning?
- Source of DNA 2. Choice of vectors 3. Restriction enzymes 4. DNA ligase 5. Host cell
What is the source of DNA in cloning?
The DNA to be cloned.
What is the role of vectors in cloning?
To carry, maintain, and replicate the cloned gene in the host cell.
What is the function of restriction enzymes?
To cut DNA.
What is the function of DNA ligase?
To join foreign DNA and vector DNA.
What is recombinant DNA?
DNA formed by joining foreign DNA and vector DNA.
What is the role of the host cell in cloning?
The recombinant DNA replicates in the host cell.
What is a plasmid?
An extra set of DNA not part of the bacterial genome.
In what organisms are plasmids common?
Bacteria.
What is the role of recombinant plasmids in molecular biology?
They are used as vectors in DNA cloning.
What is template DNA?
The source DNA containing the gene or fragment of interest.
Why is vector choice important?
Poor vector choice can cause cloning failure.
What do restriction enzymes recognize?
Specific DNA recognition sequences.
What does ligase do during cloning?
Seals or connects DNA fragments together.
What important components are usually found in plasmids?
- Marker 2. Origin of replication 3. Termination site 4. Multiple Cloning Site (MCS)
What is found in the Multiple Cloning Site (MCS)?
Recognition sequences for restriction enzymes.
What type of sequences are restriction enzyme recognition sites?
Palindromic sequences.
What recognition sequence is recognized by EcoRI?
GAATTC.
What happens when EcoRI cuts DNA?
Sticky overhangs are produced.
Why must the vector contain the same restriction site as the source DNA?
To allow compatible cutting and annealing.
What are the steps in the basic cloning process?
- Analyze the gene sequence 2. Select the restriction enzyme 3. Cut the template DNA 4. Select the vector 5. Cut the vector 6. Formation of sticky ends 7. Annealing of fragments 8. Ligation 9. Formation of recombinant plasmid 10. Introduction into host cell 11. Expression of the gene 12. Product harvesting
What happens during annealing of fragments?
Complementary sticky ends bind together.
What happens during ligation?
Ligase seals the gaps and reestablishes DNA bonds.
What is formed after ligation?
A recombinant plasmid.
What happens after the recombinant plasmid is introduced into the host cell?
The host cell produces the product encoded by the inserted gene.
What commercial product example was mentioned?
Synthetic insulin.
What are sticky overhangs?
Overhangs produced by some restriction enzymes.
Which restriction enzyme example produces sticky overhangs?
EcoRI.
What are blunt cuts?
DNA cuts without sticky overhangs.
Which enzymes mentioned produce blunt cuts?
- SmaI 2. KpnI
Does the source DNA need to come from E. coli?
No, any gene of interest can serve as the template DNA.
How is vector compatibility determined?
Based on the chosen gene fragment.
What are the types of source DNA for cloning?
- Genomic DNA 2. cDNA 3. Amplified DNA 4. Synthetic DNA
What is genomic DNA?
DNA extracted from cells and purified.
From what organisms can genomic DNA come?
Any organism.
What must first be done to genomic DNA before cloning?
The DNA must first be extracted and isolated.
What examples of genomic DNA sources were mentioned?
- Bacterial DNA 2. Human DNA
What is cDNA?
DNA produced through reverse transcription.
What is used as the template during reverse transcription?
Transcripts.
What is amplified DNA?
DNA amplified using Polymerase Chain Reaction (PCR).
Can PCR products be used directly for cloning?
Yes, the PCR product itself may be used for cloning.
What may need to be repeated if PCR does not work properly?
DNA extraction.
What is synthetic DNA?
DNA made artificially using a machine.
Is synthetic DNA commercially available?
Yes.
What is the general principle for source DNA in cloning?
As long as the material is DNA, it can become a source DNA for cloning.
What terms are often used interchangeably with gene cloning?
- Genetic engineering 2. Recombinant DNA technology
What is the basic principle of gene cloning, genetic engineering, and recombinant DNA technology?
Joining together DNA from different sources or organisms to form a recombinant DNA molecule.
What happens after recombinant DNA is formed?
It is introduced into a host cell, usually bacteria.
What does the host cell do with recombinant DNA?
The host cell replicates many copies of the recombinant DNA molecule.
What can the host cell sometimes be asked to do?
Use the genetic information in the recombinant DNA to make proteins.
What are the general steps involved in recombinant DNA technology?
- Obtaining DNA from a source 2. Joining the DNA fragment into a vector 3. Introducing the vector into a host 4. Allowing the host to produce the product of the gene 5. Harvesting the product
What gene was used as an example in the lecture?
The insulin gene.
Why is producing large amounts of insulin important?
Insulin is important for people with diabetes.
How is the insulin gene amplified?
Using PCR.
What must first be done to the vector before inserting the gene?
The vector must be cut through restriction digestion.
What does restriction digestion do to the vector?
It linearizes the vector.
What happens after the vector is linearized?
Amplified copies of the target gene are introduced into the vector.
What is the role of ligase in recombinant DNA technology?
Ligase seals the DNA fragments.
What happens to the plasmid after ligation?
The plasmid becomes circular again.
What is formed after ligation of the gene into the plasmid?
A recombinant plasmid.
How many genes does one recombinant plasmid usually carry?
One gene.
What does one recombinant plasmid typically produce?
One type of product.
What important feature do plasmids contain?
An origin of replication (ORI).
What happens to the plasmid when the host cell divides?
The plasmid is also replicated.
What is a stringent plasmid?
A plasmid that produces only a few plasmid copies during cell division.
What is a relaxed plasmid?
A plasmid that produces many plasmid copies inside the cell.