Biology 2

Chapter 19-----------------DNA TECHNOLOGY

NOTES

Recombinant DNA technology refers to the set of techniques for recombining genes from different sources in vitro and transferring this recombinant DNA into a cell where it may be expressed.

• These techniques were first developed around 1975 for basic research in bacterial molecular biology, but this technology has also led to many important discoveries in basic eukaryotic molecular biology.

• Such discoveries resulted in the appearance of the biotechnology industry. Biotechnology refers to the use of living organisms or their components to do practical tasks such as:

=> The use of microorganisms to make wine and cheese.

=> Selective breeding of livestock and crops.

=> Production of antibiotics from microorganisms.

=> Production of monoclonal antibodies.

Using recombinant DNA techniques, modem biotechnology is a more precise and systematic process than earlier research methods.

¨ It is also a powerful tool since it allows genes to be moved across the species barrier.

¨ Using these techniques, scientists have advanced our understanding of eukaryotic molecular biology.

¨ The human g enome project is an important application of this technology. This project's goal is to transcribe and translate the entire human genome in order to better understand the human organism.

¨ A variety of applications are possible for this technology, and the practical goal is the improvement of human health and food production.

I. DNA technology makes it possible to clone genes for basic research and commercial applications: an overview

Prior to the discovery of recombinant DNA techniques, procedures for altering the genes of organisms were constrained by the need to find and propagate desirable mutants.

• Geneticists relied on either natural processes, mutagenic radiation, or chemicals to induce mutations.

• In a laborious process, each organism's phenotype was checked to determine the presence of the desired mutation.

• Microbial geneticists developed techniques for screening mutants. For example, bacteria was cultured on media containing an antibiotic to isolate mutants which were antibiotic resistant.

Before 1975, transferring genes between organisms was accomplished by cumbersome and nonspecific breeding procedures. The only exception to this was the use of bacteria and their phages.

¨ Genes can be transferred from one bacterial strain to another by the natural processes of

transformation, conjugation or transduction.

¨ Geneticists used these processes to carry out detailed molecular studies on the structure and functioning of prokaryotic and phage genes.

¨ Ideal for laboratory experiments, bacteria and phages are relatively small, have simple genomes, and are easily propagated.

¨ Although the technique was available to grow plant and animal cells in culture, the workings of their genomes could not be examined using existing methods.

Recombinant DNA technology now makes it possible for scientists to examine the structure and function of the eukaryotic genome, because it contains several key components:

• Biochemical tools that allow construction of recombinant DNA.

• Methods for purifying DNA molecules and proteins of interest.

• Vectors for carrying recombinant DNA into cells and replicating it.

• Techniques for determining nucleotide sequences of DNA molecules.

II. The toolkit for DNA technology includes restriction enzymes, DNA vectors, and host organisms

A. Restriction Enzymes

Restriction enzymes are major tools in recombinant DNA technology.

¨ First discovered in the late 1960's, these enzymes occur naturally in bacteria where they protect the bacterium against intruding DNA from other organisms.

¨ This protection involves restriction, a process in which the foreign DNA is cut into small segments.

¨ Most restriction enzymes only recognize short, specific nucleotide sequences called recognition sequences. They only cut at specific points within those sequences.

Bacterial cells protect their own DNA from restriction through modification or methylation of DNA.

¨ Methyl groups are added to nucleotides within the recognition sequences.

¨ Modification is catalyzed by separate enzymes that recognize these same DNA sequences.

There are several hundred restriction enzymes and about a hundred different specific recognition sequences.

¨ Recognition sequences are symmetric in DNA that the same sequence of four to eight nucleotides is found on both strands, but run in opposite directions.

¨ Restriction enzymes usually cut phosphodiester bonds of both strands in a staggered manner, so that the resulting double‑stranded DNA fragments have single‑stranded ends, called sticky ends.

¨ The single‑stranded short extensions form hydrogen‑bonded base pairs with complementary single‑stranded stretches on other DNA molecules.

Sticky ends of restriction ftagments are used in the laboratory to join DNA pieces from different sources (cells or even different

organisms).

¨ These unions are temporary since they are only held by a few hydrogen bonds.

¨ These unions can be made permanent by adding the enzyme DNA ligase, which catalyzes formation of covalent phosphodiester bonds.

The outcome of this process is the same as natural genetic recombination, the production of recombinant DNA ‑ a DNA molecule carrying a new combination of genes.

B. Vectors

Most DNA technology procedures use carriers or vectors for moving DNA from test tubes back into cells.

Cloning vector = Bacterial plasmid or virus used to move DNA into cells.

¨ Two most often used types of vectors are bacterial plasmids and viruses.

¨ Restriction fragments of foreign DNA can be spliced into a bacterial plasmid without interfering with its ability to replicate within the bacterial cell. Isolated recombinant plasmids can be introduced into bacterial cells by transformation.

Bacteriophages, such as lambda phage, can also be used as vectors.

¨ The middle of the linear genome, which contains nonessential genes, is deleted by using restriction enzymes.

¨ Restriction fragments of foreign DNA are then inserted to replace the deleted area.

¨ The recombinant phage DNA is introduced into an E. coli cell.

¨ The phage replicates itself inside the bacterial cell.

¨ Each new phage particle carries the foreign DNA "passenger.

Sometimes it is necessary to clone DNA in eukaryotic cells rather than in bacteria. Under the right conditions, yeast and animal cells growing in culture can also take up foreign DNA from the medium.

¨ If the new DNA becomes incorporated into chromosomal DNA or can replicate itself, it can be cloned with the cell.

¨ Since yeast cells have plasmids, scientists can construct recombinant plasmids that combine yeast and bacterial DNA and that can replicate in either cell type.

¨ Viruses can also be used as vectors with eukaryotic cells. For example, retroviruses used as vectors in animal cells can integrate DNA directly into the chromosome.

C. Host Organisms

Bacteria are commonly used hosts in genetic engineering because:

¨ DNA can be easily isolated from and reintroduced into bacterial cells

¨ Bacterial cultures grow quickly, rapidly cloning the inserted foreign genes.

Some disadvantages to using bacterial host cells are that:

¨ Bacterial cells may not be able to use the information in a eukaryotic gene, since eukaryotes and prokaryotes use different enzymes and regulatory mechanisms during transcription and translation.

¨ Bacterial cells cannot make the posttranslational modifications required to produce some eukaryotic proteins. (e.g. addition of lipid or carbohydrate groups)

Eukaryotle cells may be used as hosts.

¨ Yeast cells can be used as hosts to overcome some of the limitations of using bacterial cells.

¨ Cultured plant and animal cells can be hosts for foreign DNA, but it is often difficult to get such cells to take up engineered DNA.

III. Recombinant DNA technology provides a means to transplant genes from one

species into the genome of another

A. Steps for Using Bacteria and Plasmids to Clone Genes

Recombinant DNA molecules are only useful if they can be made to replicate and produce

a large number of copies. A typical gene‑cloning procedure includes the following steps:

(See Campbell, Figure 19.3)

Step 1: Isolation of two kinds of DNA.

• Bacterial plasmids and foreign DNA containing the gene of interest are isolated.

• In this example, the foreign DNA is human, and the plasmid is from E coli and

has two genes:

amp that confers antibiotic resistance to ampicillm.

lacZ that codes for 13‑galactosidase, the enzyme that catalyzes the hydrolysis

of lactose

Note that the recognition sequence for the restriction enzyme used in this

example is within the lacZ gene.

Step 2: Treatment ofplasmid andforeign DNA with the same restriction enzyme.

The restriction enzyme cuts plasmid DNA at the restriction site, disrupting the

lacZ gene.

The foreign DNA is cut into thousands of fragments by the same restriction

enzyme; one of the fragments contains the gene of interest.

When the restriction enzyme cuts, it produces sticky ends on both the foreign

DNA fragments and the plasmid.

Step 3: Mixture offoreign DNA with clippedplasmids.

Sticky ends of the plasmid base pair with complementary sticky ends of foreign

DNA fragments.

Step 4: Addition of DNA ligase.

DNA ligase catalyzes the formation of covalent bonds, joining the two DNA

molecules and forming a new plasmid with recombinant DNA.

Step5: Introduction of recombinant plasmid into bacterial cells.

The naked DNA is added to a bacterial culture.

Some bacteria will take up the plasmid DNA by transformation.

Step 6: Production of multiple gene copies by gene cloning and selection process for

transformed cells.

Bacteria with the recombinant plasmid are allowed to reproduce, cloning the

inserted gene in the process.

Recombinant plasmids can be identified by the fact that they are ampicillin

resistant and will grow in the presence of ampicillin.

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Step 7: Final screeningfor transformed cells.

X‑gal, a modified sugar added to the culture medium, turns blue when hydrolyzed

by f3~galactosidase. It is used as an indicator that cells have been transformed by

plasmids containing the foreign insert.

Since the foreign DNA insert disrupts the lacZ gene, bacterial colonies that have

successfully acquired the foreign DNA fragment will be white. Those bacterial

colonies lacking the DNA insert will have a complete lacZ gene that produces f~‑

galactosidase and will turn blue in the presence of X‑gal.

B. Sources of Genes for Cloning

There are two major sources of DNA which can be inserted into vectors and cloned:

1. DNA isolated directly from an organism.

2. Complementary DNA made in the laboratory from mRNA templates.

DNA isolated directly from an organism contains all genes including the gene of interest.

Restriction enzymes are used to cut this DNA into thousands of pieces which are

slightly larger than a gene.

All of these pieces are then inserted into plasmids or viral DNA.

These vectors containing the foreign DNA are introduced into bacteria.

This produces the genomic library: thousands of DNA segments from a genome,

each of which is carried by a plasmid or phage.

One problem with this method is that eukaryotic genes of interest may be too large to

clone easily because they contain introns (noncoding regions). A solution is to make

complementary DNA (cDNA) in the laboratory, by using mRNA molecules as templates.

(See Campbell, Figure 19.5)

The enzyme reverse transcriptase (from retroviruses) is used to catalyze DNA

synthesis from an RNA template. A second DNA strand is synthesized using DNA

polymerase and the first DNA strand as a template.

Even if the gene of interest contains introns, the cDNA does not, because they have

been removed from the template during RNA processing.

Without introns, the cDNA gene is more manageable in size than the original gene

and also has the potential of being translated into protein by bacterial cells which

lack RNA‑processing capabilities.

To be transcribed in bacteria, the cDNA is attached to other DNA containing a

promoter and other essential transcription signals.

The cDNA method produces partial genomic libraries because mRNA molecules for a

particular gene usually cannot be distinguished from other RNA in a cell.

• By using cells from specialized tissues or a cell culture used exclusively for making

one gene product, the majority of mRNA produced is for the gene of interest.

• For example, most of the mRNA in precursors of mammalian erythrocytes is for the

protein hemoglobin.

3 3 8 DNA Technology

Inserting DNA Into Cells.

Which method is used to insert DNA into cells depends upon the vector and type of host

cell.

Most commonly, DNA is transferred into bacterial cells by transformation, the

absorption of DNA from the surrounding solution

Recombinant phage vectors package foreign DNA inside the protein coat and then

infect host cells.

Yeast cells can take up recombinant plasmids by transformation. They can also take

up linear DNA, which becomes incorporated into a chromosome by recombination.

There are more aggressive techniques for inserting foreign DNA into eukaryotic cells:

In electroporation, a brief electric pulse applied to a cell solution causes temporary

holes in the plasma membrane, through which DNA can enter.

With thin needles, DNA can be injected directly into a eukaryotic cell.

DNA attached to microscopic metal particles can be fired into plant cells with a gun.

D. Selection: Finding a Gene of Interest

identifying the bacterial clone containing the gene of interest is difficult regardless of

which approach (genomic library or cDNA) is used.

• Screening for a protein can be used if the clones translate the gene into a product.

Detection may be based on activity (for enzymes) or structure (using antibodies that

combine with the protein).

• Screening for the gene itself is most often used. These methods use base pairing

between the gene and a complementary sequence on another nucleic acid (RNA or

DNA) ‑ a process called hybridization.

Nucleic acid segments can be synthesized and used to identify a gene if a part of

the gene's nucleotide sequence is known or inferred by its protein product.

=:> The synthesized nucleic acid used to find the gene of interest is called a probe.

=> The location of a probe, which will hydrogen bond to a specific gene, is traced

by labeling it with a radioactive isotope or fluorescent tag. (See Campbell,

Figure 19.6)

• When a bacterial clone carrying the gene of interest is identified, the gene can be

easily isolated in large amounts and used for further studies or be used as probes

themselves to identify similar or identical genes.

E. Achieving Expression of Cloned Genes

Yeast genes, as well as those of more complex eukaryotes, can be expressed in bacteria.

This possibility surprised scientists because:

Prokaryotes have different signals that control gene transcription and translation.

The enzymes that recognize these signals are also different.

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Bacteria are the organisms of choice for large scale, commercial production of gene

products, because:

• They can be grown rapidly and cheaply in large fermenters.

• Their genomes are relatively simple and easy to manipulate.

• Bacteria can be manipulated to secrete a protein as it is made, simplifying

purification of the protein.

Expression of eukaryotic genes in bacterial cells can be maximized by several genetic and

biochemical methods. They include:

• Using plasmid vectors that produce many copies per cell.

• Changing the promoter controlling the gene's expression to a highly active one.

• Attaching a eukaryotic gene to the initial portion of a bacterial gene for a protein that

is usually produced in large quantities.

Eukaryotic cells must be used to study eukaryotic mechanisms of gene function and

regulation.

• While eukaryotic genes are initially cloned in bacteria, they are later returned to

eukaryotic cells such as yeast for expression and study.

• Yeast have advantages for use in the production of gene products (commercially or

for research).

1. They are easy to grow.

2. They are very simple eukaryotes.

3. It is simple to reinsert genes into yeast since they can be made to take up

DNA by transformation and have plasmids for the process.

Bacteria and yeast are not suitable for every purpose. For certain applications, plant or

animal cell cultures must be used.

Cells of more complex eukaryotes carry out certain biochemical processes not found

in yeast (e.g. only animal cells produce antibodies).

IV. Additional methods for analyzing and cloning nucleotide sequences increase the

power of DNA technology

A. Gel Electrophoresis

Gel electrophoresis is used to separate either nucleic acids or proteins based upon

molecular size, charge and other physical properties. Using this technique:

Viral DNA, plasmid DNA, and segments of chromosomal DNA can be identified by

their characteristic banding patterns after being cut with various restriction enzymes.

DNA fragments containing genes of interest can be isolated, purified and then

recovered from the gel with full biological activity.

340 DNA Technology

B. DNA Synthesis and Sequencing

It is possible to synthesize artificial genes in the laboratory (e.g. genes for insulin).

• This once tedious task can now be done with machines that can rapidly produce

genes several hundred nucleotides long.

• This approach can only be used for short genes with known nucleotide sequences.

The most common method for determining the sequence of a DNA molecule is the Sanger

Method. (See Campbell, Methods Box, for a description of DNA sequencing by the

Sanger Method.) The method is based on:

Use of restriction enzymes to cut DNA into discrete, reproducible fragments with

unique sequences.

Synthesizing in vitro DNA strands complementary to a strand from the restriction

fragment being sequenced.

Incorporation of a modified nucleotide that blocks further DNA synthesis

(dideoxyribonucleotide, which lacks 2 OH groups).

Restriction enzymes are used to cut the DNA into restriction fragments. Each fragment is

processed in the following manner:

Step 1: A preparation containing one of the strands of the DNA fragment is

divided into four portions.

Each portion is incubated with a radioactively labeled primer, DNA

polymerase, and the four deoxyribonucleotide triphosphates (ingredients

necessary for complementary strand synthesis).

Each mixture also contains one of the four nucleotides in the modified

dideoxy (dd) form.

Step 2: New strand synthesis begins with the radioactive primer and continues

until a dideoxyribonucleotide is incorporated, where synthesis stops.

• Since both deoxy and dideoxy forms of one nucleotide are present, they

compete for positions.

• A set of radioactive strands of varying lengths are eventually produced.

Step 3: The new DNA strands are separated by polyacrylamide gel electrophoresis,

which can separate strands differing by one nucleotide in length.

The sequence in the new strands is then read from the radioactive bands, and

the sequence of the original template is deduced.

DNA sequencing techniques have enabled scientists to collect thousands of DNA

sequences in computer data banks.

This knowledge greatly improves the understanding of genes and genetic control

elements.

Using a computer, scientists can scan long sequences for shorter sequences known to

be protein recognition sites and control sequences. They can also scan for

similarities to known sequences of other genes or other organisms.

Nucleotide sequences can also be automatically translated into amino acid

sequences.

DNA Technoiogy 341

C. The Polymerase Chain Reaction (PCR)

PCR is another promising new technique which allows any piece of DNA to be quickly

amplified (copied many times) in vitro. (See Campbell, Methods Box, page 380)

DNA is incubated under appropriate conditions with special primers and DNA

polymerase molecules.

Billions of copies of the DNA are produced in Just a few hours.

PCR is highly specific; primers determine the sequence to be amplified.

Only minute amounts of DNA are needed.

PCR is presently being applied in many ways for analysis of DNA from a wide variety of

sources:

• Ancient DNA fragments from a woolly mammoth; DNA is a stable molecule and

can be amplified by PCR from sources thousands, even millions, of years old.

• DNA from tiny amounts of tissue or semen found at crime scenes.

• DNA from single embryonic cells for prenatal diagnosis.

• DNA of viral genes from cells infected with difficult to detect viruses such as I‑IIV.

Amplification of DNA by PCR is being used in the hurnan genome project to produce

linkage maps without the need for large family pedigree analysis.

• DNA from sperm of a single donor can be amplified to analyze the immediate

products of meiotic recombination.

• This process eliminates the need to rely on chance that offspring will be produced

with a particular type of recombinant chromosome.

It makes it possible to study genetic markers that are extremely close together.

D. Hybridization

The technique of hybridization is used to determine the presence of a specific nucleotide

sequence.

Labeled probes complementary to the gene of interest are allowed to bind to DNA

frorn cells being tested.

Variations of this technique allows researchers to:

=> determine if a gene is present in various organisms.

=> determine not only whether a sequence is present, but how many sequences there

are and the size of the restriction fragments containing these sequences

(Southern hybridization). (See Campbell's Methods Box.)

determine whether a gene is made into mRNA, how much of that mRNA is

present, and whether the amount of that mRNA changes at different stages of

development or in response to certain regulatory signals. (Northern

hybridization)