Chapter 20: DNA Chapter 20: DNA TechnologyTechnology

Important Terminology: Important Terminology: Recombinant DNA: DNA in which nucleotide sequences Recombinant DNA: DNA in which nucleotide sequences

from 2 different sources (can be from different species) from 2 different sources (can be from different species) are combined into the same DNA molecule.are combined into the same DNA molecule.

Genetic engineering: The direct manipulation of genes Genetic engineering: The direct manipulation of genes for practical purposes (such as producing needed for practical purposes (such as producing needed proteins) – often relies on making recombinant DNA.proteins) – often relies on making recombinant DNA.

Biotechnology: The manipulation of organisms or their Biotechnology: The manipulation of organisms or their components to make useful products (modify genes and components to make useful products (modify genes and move them between organisms giving the organisms the move them between organisms giving the organisms the ability to make a new protein).ability to make a new protein).

In vitro: refers to procedures conducted OUTSIDE of a In vitro: refers to procedures conducted OUTSIDE of a living organism. living organism.

Gene Cloning: preparing gene-sized pieces Gene Cloning: preparing gene-sized pieces of DNA in multiple identical copies.of DNA in multiple identical copies.

Uses:Uses: To make many copies of a gene (could then be To make many copies of a gene (could then be

transferred to other organisms).transferred to other organisms).

To produce a protein product.To produce a protein product.

Made possible by discovery of restriction Made possible by discovery of restriction enzymes.enzymes.

These are naturally occurring enzymes in These are naturally occurring enzymes in bacteria that cut up foreign DNA.bacteria that cut up foreign DNA.

Very specific – each restriction enzyme only Very specific – each restriction enzyme only “recognizes” a particular DNA sequence – “recognizes” a particular DNA sequence –

a.k.a. a.k.a. restriction siterestriction site - and cuts both strands at - and cuts both strands at specific points within this restriction site.specific points within this restriction site.

Therefore, copies of DNA Therefore, copies of DNA molecules always yield the same molecules always yield the same restriction fragments. restriction fragments.

Restriction fragments are double-stranded DNA Restriction fragments are double-stranded DNA fragments. Because of the action of restriction fragments. Because of the action of restriction enzymes, many will have at least one single-enzymes, many will have at least one single-stranded end – called a stranded end – called a sticky sticky end.end.

These will bond with complementary stretches These will bond with complementary stretches on other DNA molecules that were cut with the on other DNA molecules that were cut with the same restriction enzyme. Use DNA ligase to same restriction enzyme. Use DNA ligase to make bond permanent.make bond permanent.

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In this way, we make recombinant DNA: DNA In this way, we make recombinant DNA: DNA that has been spliced together from 2 different that has been spliced together from 2 different sources.sources.

Finally! Cloning a Eukaryotic Gene in a Bacterial Finally! Cloning a Eukaryotic Gene in a Bacterial PlasmidPlasmidThe following is general procedure for using bacteria The following is general procedure for using bacteria to make many copies of a gene of interest…to make many copies of a gene of interest…

The plasmid is our cloning vector: carrying The plasmid is our cloning vector: carrying foreign DNA into a cell and replicating there.foreign DNA into a cell and replicating there.

The following is ONE example of a cloning The following is ONE example of a cloning procedure. There are many variation but notice procedure. There are many variation but notice that the plasmids used contain genes that will that the plasmids used contain genes that will make the identification of bacteria containing make the identification of bacteria containing recombinant plasmids easy.recombinant plasmids easy.

The purpose of using a plasmid with The purpose of using a plasmid with ampicillin resistance: only bacteria who ampicillin resistance: only bacteria who have taken up a plasmid will be able to have taken up a plasmid will be able to grow on medium containing ampicillin. grow on medium containing ampicillin. So – you will know that those bacteria So – you will know that those bacteria have been transformed.have been transformed.

Purpose of the lacZ gene: this gene Purpose of the lacZ gene: this gene encodes for an enzyme that breaks down encodes for an enzyme that breaks down lactose & another sugar called X-gal. lactose & another sugar called X-gal. When it breaks down X-gal, it makes a When it breaks down X-gal, it makes a blue byproduct. If this gene is disrupted, it blue byproduct. If this gene is disrupted, it will not function and won’t break down the will not function and won’t break down the X-gal. So, plasmids that have taken up X-gal. So, plasmids that have taken up foreign DNA will form white colonies foreign DNA will form white colonies because this gene is disrupted, plasmids because this gene is disrupted, plasmids that have not, will form blue colonies.that have not, will form blue colonies.

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At the end of this procedure, we will At the end of this procedure, we will have many colonies of bacteria have many colonies of bacteria containing many different human DNA containing many different human DNA fragments. Now – we must identify the fragments. Now – we must identify the colony that has our gene of interest – the colony that has our gene of interest – the one that we want to make many copies one that we want to make many copies of.of.

To do this, you can look for the gene To do this, you can look for the gene itself or its protein product.itself or its protein product.

A process called nucleic acid hybridization A process called nucleic acid hybridization is used to look for gene:is used to look for gene:

If part of the nucleotide sequence is known If part of the nucleotide sequence is known (maybe by working backward from its protein), a (maybe by working backward from its protein), a complementary piece of DNA can be made that complementary piece of DNA can be made that will bind to it. This is called a nucleic acid probe.will bind to it. This is called a nucleic acid probe.

The probe can be labeled with a radioactive The probe can be labeled with a radioactive isotope or fluorescent molecule and then isotope or fluorescent molecule and then identified.identified.

Once you know which colony has your Once you know which colony has your gene of interest, you can isolate it and gene of interest, you can isolate it and allow the bacteria to replicate – this will allow the bacteria to replicate – this will produce many bacterial clones – all of produce many bacterial clones – all of which have copies of the desired which have copies of the desired human gene.human gene.

What is the practical application of this What is the practical application of this procedure? procedure? Can grow large colonies of bacteria containing Can grow large colonies of bacteria containing

gene. This gene could be used for other gene. This gene could be used for other procedures – as a probe to identify similar procedures – as a probe to identify similar genes in other organisms.genes in other organisms.

Also, if the bacteria will express the inserted Also, if the bacteria will express the inserted gene, we can harvest the desired protein gene, we can harvest the desired protein product (we do this to obtain human growth product (we do this to obtain human growth hormone, insulin, etc).hormone, insulin, etc).

The entire procedure above results in MANY The entire procedure above results in MANY plasmids containing different fragments of the plasmids containing different fragments of the original genome. These are often saved and original genome. These are often saved and referred to as a genomic library. (Can use them referred to as a genomic library. (Can use them to look for other genes of interest).to look for other genes of interest).

Read about making cDNA from mRNA present in Read about making cDNA from mRNA present in cells! You are responsible for this information!cells! You are responsible for this information!

Polymerase Chain ReactionPolymerase Chain Reaction: method : method by which any piece of DNA can be by which any piece of DNA can be quickly amplified (copied many times) quickly amplified (copied many times) without being inside a cell.without being inside a cell.

PCR PCR allows any sequence of DNA to allows any sequence of DNA to copied many times – in vitro. The use of copied many times – in vitro. The use of specific primers can also allow you to make specific primers can also allow you to make copies of a specific sequence.copies of a specific sequence.

PCRPCR (which is now usually done in a (which is now usually done in a machine) involves a cycle of reactions machine) involves a cycle of reactions that can repeat over and over to copy that can repeat over and over to copy DNA.DNA.

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Restriction Fragment AnalysisRestriction Fragment Analysis

Use gel electrophoresis to sort DNA Use gel electrophoresis to sort DNA fragments (resulting from treatment with fragments (resulting from treatment with a restriction enzyme) by size.a restriction enzyme) by size.

A sample of DNA is digested with a particular A sample of DNA is digested with a particular restriction enzyme.restriction enzyme.

The restriction fragments that are produced are The restriction fragments that are produced are placed in one end of an agarose gel.placed in one end of an agarose gel.

Electric current is applied to gel.Electric current is applied to gel. DNA has a negative charge and is therefore DNA has a negative charge and is therefore

attracted to the positive end of the gel and attracted to the positive end of the gel and moves toward it.moves toward it.

Smaller fragments move faster and farther Smaller fragments move faster and farther through the gel.through the gel.

Fragments of the same length travel the same Fragments of the same length travel the same distance and form bands in the gel that are distance and form bands in the gel that are visible when a dye is added.visible when a dye is added.

Identical DNA samples will always yield identical Identical DNA samples will always yield identical banding patterns when treated with the same restriction banding patterns when treated with the same restriction enzyme.enzyme.

Applications:Applications: Useful to compare 2 different DNA samples. Useful to compare 2 different DNA samples.

Example: can compare 2 alleles for a gene. If Example: can compare 2 alleles for a gene. If they have differences in a restriction site, they they have differences in a restriction site, they will yield different banding patterns on a gel.will yield different banding patterns on a gel.

Can also recover the DNA from a gel for use.Can also recover the DNA from a gel for use.

Can use a technique called Southern Can use a technique called Southern Blotting with gel electrophoresis to find Blotting with gel electrophoresis to find bands on the gel containing genes of bands on the gel containing genes of interest.interest. Because SO MANY bands are produced when Because SO MANY bands are produced when

gel electrophoresis is performed on eukaryotic gel electrophoresis is performed on eukaryotic DNA, you need a technique to be able to DNA, you need a technique to be able to identify differences – particularly those you are identify differences – particularly those you are interested in if you are comparing two alleles.interested in if you are comparing two alleles.

The basics:The basics: Perform gel as usual.Perform gel as usual. ““Blot” gel onto special paper that DNA from gel sticks Blot” gel onto special paper that DNA from gel sticks

to.to. ““Rinse” paper with solution containing probe Rinse” paper with solution containing probe

complementary to gene of interest.complementary to gene of interest. When exposed to film, DNA bound with probe shows When exposed to film, DNA bound with probe shows

up.up. Differences in probe binding corresponds to difference Differences in probe binding corresponds to difference

in alleles – you can see these differences as in alleles – you can see these differences as differences in banding pattern.differences in banding pattern.

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Noncoding stretches of DNA have Noncoding stretches of DNA have differences in nucleotide sequences differences in nucleotide sequences between individuals just like coding between individuals just like coding segments of DNA. segments of DNA. These differences are called These differences are called restriction restriction

fragment length polymorphismsfragment length polymorphisms or RFLPs. or RFLPs.

This is basically the same as a difference in This is basically the same as a difference in coding sequence – it can also serve as a coding sequence – it can also serve as a genetic marker for a particular location in the genetic marker for a particular location in the genome and occurs in many variations in the genome and occurs in many variations in the population and is inherited in a Mendelian population and is inherited in a Mendelian fashion.fashion.

These can be separated and analyzed using These can be separated and analyzed using gel electrophoresis and Southern blotting.gel electrophoresis and Southern blotting.

This helps in mapping the human genome and This helps in mapping the human genome and in making linkage maps. in making linkage maps.

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Mapping a Genome (figuring out the Mapping a Genome (figuring out the sequence)sequence)

Constructing linkage maps using Constructing linkage maps using recombination frequencies was first step.recombination frequencies was first step.

Sequencing DNA using the Sanger Method Sequencing DNA using the Sanger Method (page 397)(page 397) The following animation shows the Sanger The following animation shows the Sanger

method but with a little more automation as it is method but with a little more automation as it is done now. It’s the same basic idea.done now. It’s the same basic idea.

This method uses DNA replication and This method uses DNA replication and special nucleotides. These special nucleotides. These nucleotides stop replication whenever nucleotides stop replication whenever they are added.they are added.

This results in many fragments of varying This results in many fragments of varying lengths. When these fragments are lengths. When these fragments are separated, you can read the sequence separated, you can read the sequence from gel.from gel.

http://http://www.pbs.org/wgbh/nova/genome/sequenwww.pbs.org/wgbh/nova/genome/sequencer.htmlcer.html

Studying Gene Expression (You Studying Gene Expression (You must read pages 399 & on!)must read pages 399 & on!)

DNA sequencing can identify regions that DNA sequencing can identify regions that are protein coding genes (presence of are protein coding genes (presence of promoters, etc) but how do we figure out promoters, etc) but how do we figure out what proteins these code for?what proteins these code for?

In vitro mutagenesis: mutations are In vitro mutagenesis: mutations are introduced into a cloned gene – reinsert it introduced into a cloned gene – reinsert it into a cell and see how phenotype is into a cell and see how phenotype is altered – may help determine the type of altered – may help determine the type of protein coded for.protein coded for.

Use DNA microarray assays to Use DNA microarray assays to determine which genes are being determine which genes are being expressed in a particular cell.expressed in a particular cell.

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(You need to watch the animation and then read (You need to watch the animation and then read this). this). cDNA is DNA that is made from mRNA.cDNA is DNA that is made from mRNA.

Genes that are being expressed are the only Genes that are being expressed are the only genes in a cell making mRNA. genes in a cell making mRNA.

Scientists can isolate mRNA from cells and use Scientists can isolate mRNA from cells and use reverse transcriptase to produce DNA that must reverse transcriptase to produce DNA that must have coded for the mRNA – this is what is called have coded for the mRNA – this is what is called cDNA and what is referred to in the microarray cDNA and what is referred to in the microarray animation.animation.

Applications of DNA technologyApplications of DNA technology

Determine evolutionary relationships by Determine evolutionary relationships by comparing genome sequences.comparing genome sequences.

Can also study the variation within our species. Can also study the variation within our species. We are 99.9% the same as all other humans We are 99.9% the same as all other humans (our evolutionary history is pretty short). We can (our evolutionary history is pretty short). We can look at differences in genes and relate them to look at differences in genes and relate them to human evolution/migratory patterns.human evolution/migratory patterns.

Diagnosis of DiseaseDiagnosis of Disease Can identify abnormal alleles for early Can identify abnormal alleles for early

identification of genetic diseases.identification of genetic diseases.

Look for HIV RNA in blood and tissue samplesLook for HIV RNA in blood and tissue samples

Gene TherapyGene Therapy Altering a person’s genes (give someone with an Altering a person’s genes (give someone with an

abnormal gene the normal gene)abnormal gene the normal gene) In people with disorders traceable to a single defective In people with disorders traceable to a single defective

gene, it should theoretically be possible to replace or gene, it should theoretically be possible to replace or supplement the defective gene with a normal allele. The supplement the defective gene with a normal allele. The new allele could be inserted into the somatic cells of the new allele could be inserted into the somatic cells of the tissue affected by the disorder.tissue affected by the disorder.

Very little scientifically strong evidence of effectiveness. Very little scientifically strong evidence of effectiveness. Even when genes are successfully and safely Even when genes are successfully and safely transferred, their activity typically diminishes after a short transferred, their activity typically diminishes after a short period.period.

Technical questions:Technical questions:

How can the activity of the transferred gene How can the activity of the transferred gene be controlled so that cells make appropriate be controlled so that cells make appropriate amounts of the gene product at the right time amounts of the gene product at the right time and in the right place?and in the right place?

How can we be sure that the insertion of the How can we be sure that the insertion of the gene doesn’t harm some other cell function?gene doesn’t harm some other cell function?

Ethical/Social QuestionsEthical/Social Questions Will it inevitably lead to eugenics (a deliberate Will it inevitably lead to eugenics (a deliberate

effort to control the genetic makeup of the human effort to control the genetic makeup of the human population)?population)?

Should we try to treat human germ-line cells in the Should we try to treat human germ-line cells in the hope of correcting a defect in future generations? hope of correcting a defect in future generations? Should we interfere with evolution in this way? Should we interfere with evolution in this way? The elimination of unwanted alleles from the gene The elimination of unwanted alleles from the gene pool could backfire – genes that are damaging pool could backfire – genes that are damaging under some conditions may be advantageous under some conditions may be advantageous under other conditions.under other conditions.

Pharmaceutical Pharmaceutical ProductsProducts – using – using cloning techniques cloning techniques to make insulin, to make insulin, growth hormone, growth hormone, etc. We insert the etc. We insert the genes that make genes that make these proteins into these proteins into other organisms so other organisms so that we can harvest that we can harvest the protein and give the protein and give it to people.it to people.

Environmental UsesEnvironmental Uses Engineer microbes that Engineer microbes that

can break down heavy can break down heavy metals, toxins in mining metals, toxins in mining sites, oil spills.sites, oil spills.

AgricultureAgriculture Insert human genes into farm animals that produce the Insert human genes into farm animals that produce the

protein to harvest – some secrete the desired protein in protein to harvest – some secrete the desired protein in their milk. their milk.

Can make organisms that produce better wool, meat, etc.Can make organisms that produce better wool, meat, etc. Plants with resistance to pesticides and improved Plants with resistance to pesticides and improved

nutritional value (rice containing beta carotene needed to nutritional value (rice containing beta carotene needed to make vitamin A)make vitamin A)

MIT students working with E. coli bacteria inserted a gene into the bacteria that made them smell like mint – just to make their working environment more pleasant.

Forensic EvidenceForensic Evidence Comparison of samples Comparison of samples

of body fluids/tissues at of body fluids/tissues at crime scene with crime scene with suspects.suspects.

Use probes for RFLPs Use probes for RFLPs and gel electrophoresis and gel electrophoresis – provides an – provides an individual’s DNA individual’s DNA fingerprint. Likelihood fingerprint. Likelihood that banding pattern on that banding pattern on gel will be the same for gel will be the same for different people is VERY different people is VERY small because RFLPs small because RFLPs are inherited & variable.are inherited & variable.