biotechnology

CloningCloning

Recap What is cloning?

Generating identical copies of organisms, cells, or replicating nucleic acid sequences from organisms

Dolly (the sheep) is a clone, but a natural identical twin is not a clone.

DNA sequence amplified by growth is a clone,

How to clone something?

Application

Recap What is cloning?

Generating identical copies of organisms, cells, or replicating nucleic acid sequences from organisms

Dolly (the sheep) is a clone, but a natural identical twin is not a clone.

DNA sequence amplified by growth is a clone,

How to clone something?

Application

Cloning Specific GenesCloning Specific Genes1. Insert the DNA into vector

Gene of interest is inserted into small DNA molecules known as plasmids, which are self-replicating, extrachromosomal genetic elements originally isolated from the bacterium, Escherichia coli. Vectors

Plasmids, or phage

The circular plasmid DNA is opened using the same endonuclease that was used to cleave the genomic DNA. 

2. The inserted DNA is joined to the plasmid DNA using another enzyme, DNA ligase, to give a recombinant DNA molecule.

The new plasmid vector contains the original genetic information for replication of the plasmid in a host cell plus the inserted DNA

3.The cell E.coli, yeast

1. Insert the DNA into vector

Gene of interest is inserted into small DNA molecules known as plasmids, which are self-replicating, extrachromosomal genetic elements originally isolated from the bacterium, Escherichia coli. Vectors

Plasmids, or phage

The circular plasmid DNA is opened using the same endonuclease that was used to cleave the genomic DNA. 

2. The inserted DNA is joined to the plasmid DNA using another enzyme, DNA ligase, to give a recombinant DNA molecule.

The new plasmid vector contains the original genetic information for replication of the plasmid in a host cell plus the inserted DNA

3.The cell E.coli, yeast

Plasmid vector

DNA fragment to be cloned

Vectors Vectors

The substance that can serve as carriers to allow replication of recombinant DNAs.

Plasmids

Phage

The substance that can serve as carriers to allow replication of recombinant DNAs.

Plasmids

Phage

PlasmidsPlasmids

Double stranded circles of DNA that can replicate autonomously.

Multiple cloning site. The place where foreign DNA fragmentscan be inserted.

Double stranded circles of DNA that can replicate autonomously.

Multiple cloning site. The place where foreign DNA fragmentscan be inserted.

MCS

Phage λPhage λ

A phage λ virion has a head, which contains the viral DNA genome, and a tail, which functions in infecting E.coli host cells.

Advantages over plasmids: They infects cells much more efficiently than plasmids transform cells. The yield of clones with vectors usually higher.

A phage λ virion has a head, which contains the viral DNA genome, and a tail, which functions in infecting E.coli host cells.

Advantages over plasmids: They infects cells much more efficiently than plasmids transform cells. The yield of clones with vectors usually higher.

Viral Genome

The CellThe Cell

E.coli:Normal E. coli cells cannot take up plasmid DNA fro

m the medium. Exposure to high concentration of certain divalent cations, CaCl2, makes a small fraction of cells permeable to foreign DNA.

Each component cell incorporates a single plasmid DNA molecule.

E.coli:Normal E. coli cells cannot take up plasmid DNA fro

m the medium. Exposure to high concentration of certain divalent cations, CaCl2, makes a small fraction of cells permeable to foreign DNA.

Each component cell incorporates a single plasmid DNA molecule.

Inserted SequenceInserted Sequence

Source of Nucleic acid to be cloned:

-DNA directly from organism

-DNA synthesized or amplified in vitro

-Generally a specific sequence.

Source of Nucleic acid to be cloned:

-DNA directly from organism

-DNA synthesized or amplified in vitro

-Generally a specific sequence.

Two Important EnzymesTwo Important Enzymes

Restriction Enzymes: cuts the DNA from any organism at specific sequences of a

few nucleotides, generating a reproducible set of fragments.

DNA Ligases: insert DNA restriction fragments into replicating DNA mol

ecules producing recombinant DNA.

Restriction Enzymes: cuts the DNA from any organism at specific sequences of a

few nucleotides, generating a reproducible set of fragments.

DNA Ligases: insert DNA restriction fragments into replicating DNA mol

ecules producing recombinant DNA.

first recombinant DNA experiments

1971 scientists manipulated DNA and placed them into bacteria

1972 scientists joined two DNA molecules from different sources using the endonuclease EcoRI (to cut) and DNA ligase (to reseal)

1971 scientists manipulated DNA and placed them into bacteria

1972 scientists joined two DNA molecules from different sources using the endonuclease EcoRI (to cut) and DNA ligase (to reseal)

mechanismmechanism

Plasmid vector

+

DNA fragment to be cloned

Enzymatically insert DNA into plasmid vector

Recombinant plasmid

Mix E.coli cells with plasmids

Bacterial chromosome

Independent plasmid

replication

Cell multiplication

How?How?

Introduce the new vector into host The new vector is inserted back into a host where many copies of the genetic sequence are made as the cell grows and divide with the replicating vector inside. Isolate the newly-synthesized DNA or the protein coded for by the inserted gene.

The host may even transcribe and translate the gene and obligingly produce product of the inserted gene.

Alternatively, many copies of the DNA gene itself may be isolated for sequencing the nucleic acid or for other biochemical studies.

cloning DNAcloning DNA

Library ConstructionLibrary Construction

A library is a collection of different cloned DNAs from a single source.

Genomic library – for genome sequencing

cDNA library – derived from mRNA of a particular tissue, for isolating specific genes

A library is a collection of different cloned DNAs from a single source.

Genomic library – for genome sequencing

cDNA library – derived from mRNA of a particular tissue, for isolating specific genes

Revolution in cloning: Polymerase Chain Reaction

(PCR)--1986

What is the PolymeraseChain Reaction?

What is the PolymeraseChain Reaction?

It’s a means of selectively amplifying a particular segment of DNA.

The segment may represent a small part of a large and complex mixture of DNAs:e.g. a specific exon of a human gene.

It can be thought of as a molecular photocopier.

It’s a means of selectively amplifying a particular segment of DNA.

The segment may represent a small part of a large and complex mixture of DNAs:e.g. a specific exon of a human gene.

It can be thought of as a molecular photocopier.

How Powerful is PCR?How Powerful is PCR?

PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours.

The template DNA need not be highly purified — a boiled bacterial colony.

The PCR product can be digested with restriction enzymes, sequenced or cloned.

PCR can amplify a single DNA molecule, e.g. from a single sperm.

PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours.

The template DNA need not be highly purified — a boiled bacterial colony.

The PCR product can be digested with restriction enzymes, sequenced or cloned.

PCR can amplify a single DNA molecule, e.g. from a single sperm.

PCRPCR

Polymerase Chain Reaction (PCR) (PCR cycle)1. DNA amplified by heating to break hydrogen

bonds, yielding single stranded DNA2. Short nucleotide sequences act as primers for

DNA replication added3. Enzyme, DNA polymerase, begins at primers and

synthesizes a DNA strand complementary to the region between the primers, a process called "primer extension"

4. Example: 10 cycles = 1,024 copies, 30 cycles = 1,073,741,820 copies

Polymerase Chain Reaction (PCR) (PCR cycle)1. DNA amplified by heating to break hydrogen

bonds, yielding single stranded DNA2. Short nucleotide sequences act as primers for

DNA replication added3. Enzyme, DNA polymerase, begins at primers and

synthesizes a DNA strand complementary to the region between the primers, a process called "primer extension"

4. Example: 10 cycles = 1,024 copies, 30 cycles = 1,073,741,820 copies

Can I PCR Amplify RNA?Can I PCR Amplify RNA?

Not directly — the DNA polymerase requires a DNA template and will not copy RNA.

mRNA can first be copied into cDNA using reverse transcriptase.

cDNA is a template for PCR — it need not be double-stranded.

Not directly — the DNA polymerase requires a DNA template and will not copy RNA.

mRNA can first be copied into cDNA using reverse transcriptase.

cDNA is a template for PCR — it need not be double-stranded.

PCR AnimationPlease click here.

1st cycle 2nd cycle 3rd cycle

Process

Denature

Anneal Primer

Replicate DNA

Applications of PCRApplications of PCR

Mutation testing, e.g. cystic fibrosis. Diagnosis or screening of acquired diseases,

e.g. AIDS. Genetic profiling in forensic, legal and bio-

diversity applications. Quantitation of mRNA in cells or tissues.

Mutation testing, e.g. cystic fibrosis. Diagnosis or screening of acquired diseases,

e.g. AIDS. Genetic profiling in forensic, legal and bio-

diversity applications. Quantitation of mRNA in cells or tissues.

biotechnologybiotechnology

Biotechnology helps to meet our basic needs. Food, clothing, shelter, health and safety

Improvements by using science Science helps in production plants, animals and

other organisms

Also used in maintaining a good environment that promotes our well being

Biotechnology helps to meet our basic needs. Food, clothing, shelter, health and safety

Improvements by using science Science helps in production plants, animals and

other organisms

Also used in maintaining a good environment that promotes our well being

Analyzing cloned sequences

Electrophoresis1. Southern blotting (DNA)

2. Northern blotting (RNA)

3. Western blotting (protein)

First recombinant DNA experiments

Herbert Boyer discovered a new technique called gel electrophoresis to separate DNA/RNA fragments or proteins

A current is applied so that the negative charged DNA/RNA or proteins migrate towards the positive electrode and is separated by fragment size

Herbert Boyer discovered a new technique called gel electrophoresis to separate DNA/RNA fragments or proteins

A current is applied so that the negative charged DNA/RNA or proteins migrate towards the positive electrode and is separated by fragment size

Further Applications

RFLP

A Brief Tour of DNA fingerprinting

•Although the structure of DNA is the same throughout all species of plants, animals and microorganisms, each individual organism looks different.

•This is due to the order in which DNA base pairs are sequenced.

•Not only does this order make you a human rather than a dog or a daffodil, it also makes each person unique.

•Sequences of DNA differ from person to person, but every cell within the same person contains the same sequence of DNA. So, your hair, blood, skin and all of the other cells in your body are exactly the same at the molecular level.

DNA FingerprintDNA Fingerprint

This comes in very handy when police are investigating a crime. If a person left a strand of hair, a drop of blood or any other cells at a crime scene, the police will know that that person was there.

But, the human genome contains about 3 billion base pairs of DNA. Examining this large a sequence seems like it would be tedious, time-consuming and expensive, so how is it done?

This comes in very handy when police are investigating a crime. If a person left a strand of hair, a drop of blood or any other cells at a crime scene, the police will know that that person was there.

But, the human genome contains about 3 billion base pairs of DNA. Examining this large a sequence seems like it would be tedious, time-consuming and expensive, so how is it done?

DNA FingerprintDNA Fingerprint

On some human chromosomes, there are sequences of repeated DNA (9 to 80 base pairs long).

The number of repeats can vary from about one to thirty and are not the same from person to person.

These sequences are called Variable Number of Tandem Repeats (VNTRs). Within the VNTRs there are sites where an enzyme can cut the DNA, and the location of these sites also varies from person to person.

Cutting with the enzyme will lead to DNA fragments of different lengths, which are called Restriction Fragment Length Polymorphisms (RFLPs).

These DNA fragments can be separated on an agarose gel based on their size. The RFLPs can be seen by probing using complementary radioactive DNA, and they are used to compare different samples of DNA.

On some human chromosomes, there are sequences of repeated DNA (9 to 80 base pairs long).

The number of repeats can vary from about one to thirty and are not the same from person to person.

These sequences are called Variable Number of Tandem Repeats (VNTRs). Within the VNTRs there are sites where an enzyme can cut the DNA, and the location of these sites also varies from person to person.

Cutting with the enzyme will lead to DNA fragments of different lengths, which are called Restriction Fragment Length Polymorphisms (RFLPs).

These DNA fragments can be separated on an agarose gel based on their size. The RFLPs can be seen by probing using complementary radioactive DNA, and they are used to compare different samples of DNA.

DNA FingerprintDNA Fingerprint

DNA fingerprinting can be used to identify a child’s parents. Each child inherits one set of chromosomes from each parent. This is why children resemble both of their parents. A child who has a mom with brown hair and blue eyes and a dad with blond hair and brown eyes might end up with brown hair from his mom and brown eyes from his dad. RFLPs are inherited in the same way, some from the mother and some from the father.

DNA fingerprinting can be used to identify a child’s parents. Each child inherits one set of chromosomes from each parent. This is why children resemble both of their parents. A child who has a mom with brown hair and blue eyes and a dad with blond hair and brown eyes might end up with brown hair from his mom and brown eyes from his dad. RFLPs are inherited in the same way, some from the mother and some from the father.

RFLPRFLP

In this example, a family consists of a mom and dad, two daughters and two sons. The parents have one daughter and one son together, one daughter is from the mother’s previous marriage, and one son is adopted, sharing no genetic material with either parent. After amplifying the VNTR DNA from each member of the family, it is cut with a restriction enzyme and run on an agarose gel.

In this example, a family consists of a mom and dad, two daughters and two sons. The parents have one daughter and one son together, one daughter is from the mother’s previous marriage, and one son is adopted, sharing no genetic material with either parent. After amplifying the VNTR DNA from each member of the family, it is cut with a restriction enzyme and run on an agarose gel.

DNA FingerprintDNA Fingerprint

•The police use the same analysis to determine the identity of a person at a crime scene. After collecting a DNA samples from the scene and any suspects, the police amplify the VNTRs and digest the DNA with a restriction enzyme. •The samples are run on an agarose gel, and the bands found at the crime scene are aligned with those of the suspects’. •DNA fingerprints can do two things, they can either prove someone’s innocence, or prove their guilt.

The next example shows how DNA fingerprinting can point to a criminal. DNA samples were taken from a crime scene, the female victim and two suspects in a sexual assault case. The victim’s boyfriend was also tested. The DNA ladders are used to judge the sizes of the DNA fragments. Control samples are also run, to ensure that the experiment is done correctly.

production

products of biotech

Agriculture 1. Improved Nutritional Quality

Milled rice is the staple food for a large fraction of the world's human population. Milling rice removes the husk and any beta-carotene it contained. Beta-carotene is a precursor to vitamin A, so it is not surprising that vitamin A deficiency is widespread, especially in the countries of Southeast Asia.

The synthesis of beta-carotene requires a number of enzyme-catalyzed steps. In January 2000, a group of European researchers reported that they had succeeded in incorporating three transgenes into rice that enabled the plants to manufacture beta-carotene in their endosperm.

2. Insect Resistance. Bacillus thuringiensis is a bacterium that is pathogenic for a number of

insect pests. Its lethal effect is mediated by a protein toxin it produces. Through recombinant DNA methods, the toxin gene can be introduced directly into the genome of the plant where it is expressed and provides protection against insect pests of the plant.

Agriculture 1. Improved Nutritional Quality

Milled rice is the staple food for a large fraction of the world's human population. Milling rice removes the husk and any beta-carotene it contained. Beta-carotene is a precursor to vitamin A, so it is not surprising that vitamin A deficiency is widespread, especially in the countries of Southeast Asia.

The synthesis of beta-carotene requires a number of enzyme-catalyzed steps. In January 2000, a group of European researchers reported that they had succeeded in incorporating three transgenes into rice that enabled the plants to manufacture beta-carotene in their endosperm.

2. Insect Resistance. Bacillus thuringiensis is a bacterium that is pathogenic for a number of

insect pests. Its lethal effect is mediated by a protein toxin it produces. Through recombinant DNA methods, the toxin gene can be introduced directly into the genome of the plant where it is expressed and provides protection against insect pests of the plant.

applications

Agriculture

3. Disease Resistance. Genes that provide resistance against plant viruses have been

successfully introduced into such crop plants as tobacco, tomatoes, and potatoes

4. Herbicide Resistance. Genes for resistance to some of the newer herbicides have been introduced into

some crop plants and enable them to thrive even when exposed to the weed

killer.

Agriculture

3. Disease Resistance. Genes that provide resistance against plant viruses have been

successfully introduced into such crop plants as tobacco, tomatoes, and potatoes

4. Herbicide Resistance. Genes for resistance to some of the newer herbicides have been introduced into

some crop plants and enable them to thrive even when exposed to the weed

killer.

applications

Medicine

Development of novel therapeutic molecules for medical treatments

Drug delivery systems smart drugs for cancer and autoimmune diseases

gene-based diagnostics and therapies pharmaco-genomics and personalised medicine health and longevity

Medicine

Development of novel therapeutic molecules for medical treatments

Drug delivery systems smart drugs for cancer and autoimmune diseases

gene-based diagnostics and therapies pharmaco-genomics and personalised medicine health and longevity

applications

public reaction

However, concerns have focused on both applications and ethical implications: Gene therapy experiments have raised the

question of eugenics (artificial human selection) as well as testing for diseases currently without a cure

In agriculture, there is concern about gene containment and the creation of “super weeds” (herbicide and/or pesticide resistant weeds)

Today, fears have focused on genetically engineered foods in the marketplace and has resulted in the rapid growth of the organic food industry

However, concerns have focused on both applications and ethical implications: Gene therapy experiments have raised the

question of eugenics (artificial human selection) as well as testing for diseases currently without a cure

In agriculture, there is concern about gene containment and the creation of “super weeds” (herbicide and/or pesticide resistant weeds)

Today, fears have focused on genetically engineered foods in the marketplace and has resulted in the rapid growth of the organic food industry