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There once was a small black-and white-striped fish (that might even be in your aquarium) that was being studied by Dr. Mark Keating of Harvard. And Dr. Mark asked the question, “if the zebrafish can regenerate fins and eye parts, could it regenerate. . . . ?”

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So he took this 1-inch long fish and cut away about 20% of their two chambered hearts. The incisions through the abdomen were blotted to stop bleeding, the fish were returned to the water and 8 out of 10 survived the experiment. “They sort of hang out at the bottom of the tank” it was reported.

But within 10 days, the fish began to swim normally and were healthy as nonexperimental fish.

After 2 months, the fish had totally regenerated their hearts, replacing all the tissue and the cells were vigorously beating with little or no scarring of the tissue.

Why should we care??

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DNA TECHNOLOGY MAKES IT POSSIBLE TO

CLONE GENES FOR BASIC RESEARCH AND

COMMERCIAL APPLICATIONS

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How do you (or did you) use a bacterial plasmid to clone a gene?

1) Use of restriction enzymes

2) Mix the cut plasmid and source of the gene

-jellyfish in the pGLO lab

-but what if it was a heart muscle repair gene?

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3) What occurs during the mixing of the cut plasmid and the cut DNA of interest?

4) But how do you know that recombination has even occurred?

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5) All you have at this point is, hopefully, plasmid with a new gene inserted.

6) Introduce this modified plasmid into bacteria.

7) How will we select for the recombinant bacteria? That is, how will you locate them? How will you know which ones have taken up the plasmid and which ones haven’t

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8) Ways to distinguish the recombined bacteria:

-antibiotic resistance

-reporter gene or marker such as GFP

-if you were isolated the heart muscle tissue repair gene you could use GFP

-some kind of fluorescence

9) Select those bacteria that have been transformed

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10) But you have to have lots of the product of this gene so you must let these bacteria reproduce.

11) So this zebrafish gene that is responsible for repairing torn heart muscle tissue might be in humans or could be implanted in human cardiac cells.

12) How do find out if we even already have this gene but we are just not “using” it?

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13) By sequencing the gene of the zebrafish and comparing its sequence to the database for our genome.

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What has this procedure contributed to genetics?

1) Study gene products

2) Produce necessary proteins: insulin, human growth hormone, receptor proteins

3) Produce transgenic organisms: rice that will produce levels of vitamin A for those people / areas deficient in vit. A

4) Study gene regulation or gene structure

5) Develop a gene library

6) Amplify a particular gene of interest

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Dideoxy Method of DNA Sequencing

1) A modified form of the normal deoxyribonucleotide triphosphate (dNTP) is used.

2) The modified form is called a dideoxy- because its 3’ hydroxyl is removed which prevents elongation of the DNA strand.

3) These dideoxy’s are also tagged with a particular molecule that when exposed to a laser will be excited and emit a different color.

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4) SS DNA of interest (lots of copies) is/are isolated and mixed with:

-dNTPs

-DNA primer

-DNA polymerase

-Buffer

-fluorescent-tagged ddNTPs

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5) The gene of interest is then copied but whenever a ddNTP is added to the growing strand, replication stops.

6) This will produce mixture of different sized fragments that are complementary to the gene of interest.

7) The exact lengths can be used to determine the position of each base in the growing chain.

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8) At this point you have fragments of ds DNA and the fluorescently tagged bases are complementary to your gene.

9) Heat up the DNA, separate the strands; electrophorese the different length strands (capillary electrophoresis)

10) As the fragments run off the gel, a laser excites the different tag on the base and emits a different color for each base.

11) This color and therefore base is recorded.

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12) Remember, we have just determined the order of the bases in the complementary strand so the actual gene is complementary to the readout.

13) Dye Sequencing Animation from CSHL

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Another method to clone a gene is by the polymerase chain reaction

What were the requirements needed to perform this technique?

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What was PCR’s contribution to genetics?

1) The ability to make millions of copies of a gene

2) Amplifies a very specific region: PCR Animation

3) Use in forensics

-let’s let the specific region being amplified be amplified in different amounts in various individuals. A region could be present as 1 “repeat” or 4 “repeats” or 40 “repeats.” So the region is highly variable.

-and now let’s say we have 5 to 10 different regions where various kinds of repeats could be located.

-the odds that two random individuals would share this same genetic pattern by chance is about 1 in 10 billion.

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4) Its use in paternity diagnosis:

-same as in forensics

5) Its use in medical diagnosis:

-let the Alu insert be present whenever a severe disorder of a disease occurs. This “Alu insert” would be a marker for a particular disease. Statistics would determine the probably of the development of the disease when the marker (s) is present

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6) Its use in evolutionary applications

-the more closely associated DNA sequences are between organisms, the more closely related they are.

-the more specific markers, regions, genes, etc. are in common, the more associated are the organisms.

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Figure 20.1 An overview of how bacterial plasmids are used to clone genes

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Figure 20.2 Using a restriction enzyme and DNA ligase to make recombinant DNA

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Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 3)

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Figure 20.4 Using a nucleic acid probe to identify a cloned gene

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Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene

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Figure 20.6 Genomic libraries

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Figure 20.7 The polymerase chain reaction (PCR)

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Figure 20.8 Gel electrophoresis of macromolecules

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Restriction Fragment Length Patterns

RFL Patterns can be used to distinguish different alleles

Alleles are different forms of a certain gene. They can differ because of a different base sequence. This difference in base sequence then allows one form of the allele to be functional, and the other not.

Let’s say we have 2 different cloned gene samples and that the base sequence near these alleles has a restriction site or sequence. If in one of the alleles there is a different base and thus changing the restriction site, the restriction enzyme will not cut there.

This inability to cut at one of the sites will produce DNA fragments that are of different sizes / numbers.

So when the fragments from allele 1 and the fragments from allele 2 are run on a gel, different bands are produced for each of the alleles.

This shows that there is a single base difference between the two alleles of the two different DNA samples.

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Figure 20.9 Using restriction fragment patterns to distinguish DNA from different alleles

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Southern Blotting

What if we want to search for a gene of interest or a sequence of interest amongst a collection of DNA fragments?

We could “see” which fragment amongst the 100’s has our gene of interest (GOI) by attaching a radioactive probe to it. This is what a Southern Blot does, it isolates a strand of DNA by hybridization

with a radioactive probe.

Purposes of Southern Blotting:

Identifies all the fragments of DNA in your sample that have the sequence of interest.

Can be used to find sequences in noncoding sections (introns) and therefore differences / similarities between organisms.

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Figure 20.10 Restriction fragment analysis by Southern blotting

Alkaline solution draws the DNA up into the nitrocellulose paper and denatures the DNA into single strands

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RFLPs

Differences in nucleotide sequences between alleles or in the introns can create different sized fragments of DNA because these differences change the location of where a restriction enzyme can cut.

These different sized fragments are called Restriction Fragment Length Polymorphisms.

These RFLPs can serve as genetic markers telling you what your “banding pattern” looks like as compared to someone else.

Look at step 5 of the next slide

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Figure 20.10 Restriction fragment analysis by Southern blotting

Alkaline solution draws the DNA up into the nitrocellulose paper and denatures the DNA into single strands

Individuals I and II have the same genetic marker.

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Human Genome Project

It’s about:

determining the entire sequence of all 22 pairs of autosomes plus the X and Y chromosome

determining the sequence of many other species: E. coli, S. cerevisiae (yeast), Drosophila (fruit fly), Arabadopsis, mouse, C. elegans (nematode)

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Entire Genomes Can Be Mapped

Genetic Mapping

The markers or RFLPs can be used to determine recombination frequencies which will tell you how far apart they are.

These markers could also be genes

This helps put genes “in order.”

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Entire Genomes Can Be Mapped (cont’d)

The Ordering of DNA Fragments

You can do this by cutting up the whole chromosome with restriction enzymes and putting the fragments together.

You will use restriction enzymes that create some overlapping amongst the fragments

Chromosome walking: this helps to provide a series of markers along a chromosome

You first need to know something about a gene or marker and then make a probe, probe 1, to bind to its 3’end.

Take your DNA and cut it with two different restriction enzymes and clone copies of these fragments.

Expose your probe to Library II, the DNA of the cloned fragments cut with a different restriction enzyme.

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Entire Genomes Can Be Mapped (cont’d)

The Ordering of DNA Fragments (cont’d)

Chromosome walking: this helps to provide a series of markers along a chromosome

Your probe 1 will bind to a fragment of the DNA from library II.

Isolate this fragment

Make a probe, probe 2, for its 3’ end.

Expose probe 2 to the DNA from library 1 and this will bind further along the DNA, hence walking down the DNA fragment.

If you keep repeating this, you move all the way down the fragment with these probes and where these probes bind serve as sequences or markers in a known order.

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Figure 20.11 Chromosome walking

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Figure 20.12 Sequencing of DNA by the Sanger method (Layer 1)

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Figure 20.12 Sequencing of DNA by the Sanger method (Layer 2)

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Figure 20.12 Sequencing of DNA by the Sanger method (Layer 3)

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Figure 20.12 Sequencing of DNA by the Sanger method (Layer 4)

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Figure 20.13 Alternative strategies for sequencing an entire genome

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Table 20.1 Genome Sizes and Numbers of Genes

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Figure 20.14a DNA microarray assay for gene expression

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Figure 20.14b DNA microarray assay for gene expression

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Practical Applications of DNA Technology

1. Diagnosis of Diseases

a) Knowing the sequence of a genome such as that of HIV, you can determine whether or not it is present in a blood/semen/tissue sample.

b) Genes for a variety of diseases have been cloned and therefore can be identified: hemophilia, cystic fibrosis, Duchenne muscular dystrophy

c) These genetic diseases can be detected by exposing the DNA to radioactive probes that will bind to the mutant alleles.

d) RFLPs can be used to identify a marker that is associated with a genetic disorder.

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Practical Applications of DNA Technology

2. Human Gene Therapy

a) Most effective if there is a single genetic disorder.

b) Can we replace the gene or at least insert a good copy of it?

c) Insert it into the somatic cells so then then the gene product can be released into the blood stream.

3. Pharmaceutical Products

a) Human insulin

b) Human growth hormone

c) Tissue plasminogen activator: helps to dissolve blood clots and thus reduces the risk of a blood clot after a heart attack.

d) Protein products that block receptors (HIV)

e) Produce viral noninfectious protein products for vaccines

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Figure 20.16 One type of gene therapy procedure

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Practical Applications of DNA Technology

4. Forensics

a) The DNA sequence of each individual is unique and these variations produce different RFLPs.

b) Compare blood from the suspect, the victim and the crime scene to associate the suspect.

c) Compare blood of mother, child and possible father.

d) STR’s or Simple Tandem Repeats: this is where a short series of repeating bases can occur 10 to 100 times. PCR is used to selectively amplify one of these regions and will run out differently on a gel as different sized fragments distinguish you from me.

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Figure 20.17 DNA fingerprints from a murder case

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Practical Applications of DNA Technology

5. Environmental Uses

a) Microorganisms can extract toxic metals from soil

b) Bacteria can be engineered to degrade toxic organic compounds.

6. Agricultural Uses

a) Transgenic Animals: carry genes from other species

i. How is a transgenic animal made: remove eggs; fertilize them in vitro; insert the cloned, desired gene into the nuclei of the eggs; some eggs will integrate the injected DNA into the genomic DNA; gene product is expressed; these eggs are them implanted into mother; embryo develops and it has the “new” genes.

ii. Isolate a breed of cow that contains a gene that makes more muscle mass and isolate the gene, clone it and insert it into other cows.

iii. Insert a gene for blood clotting into cows and have its gene product released into the milk of the cow. Purify it.

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Practical Applications of DNA Technology

7. Genetic Engineering in Plants

a) To:

i. Delay ripening

ii. Provide frost resistance

iii. Provide pest resistance

iv. Resist spoilage

v. Vitamin A or other vitamin deficiency correction

vi. Increase nitrogen fixation by plants

vii. Create drugs / proteins for use as medicines / vaccines.

Vaccine for hepatitis B

Tobacco plants are being used to create antibiotics against bacteria that cause tooth decay.

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Figure 20.20 “Golden” rice contrasted with ordinary rice

Increase the nutritional value of food by inserting the gene to make beta-carotene. Beta-carotene makes vitamin A in our bodies so this transgenic rice could help prevent vit. A deficiencies. 70% of children under the age of 5 in Southeast Asia suffer from Vit A deficiency.

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Practical Applications of DNA Technology

b) How do you get these genes into plants?

i. By the use of a plasmid called a Ti plasmid. This plasmid comes from a bacterium that causes tumors in plants. The Ti plasmid will insert some of its DNA into the plant’s DNA causing the tumor but researchers work with a non-tumorigenic form.

ii. So give the non-tumorigenic plasmid a new gene and then infect plant cells in culture or directly into the plant cells. The plant cells then insert the new gene into their genome.

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Figure 20.19 Using the Ti plasmid as a vector for genetic engineering in plants

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Genetically Modified Organisms or GMOs

A lot of concern surrounds recombinant microbes.

1. Can new pathogens be created?

a) What if a bacterium had cancer genes inserted into it?

b) What about a flu virus getting these cancer genes?

c) Is this possible? Is there a real risk?

2. Implemented Safety Measures

a) Strict measures to prevent microbes from escaping the labs

b) Microbe strains are modified so they cannot survive outside the lab.

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Genetically Modified Organisms or GMOs (cont’d)

A GMO is any organism that has acquired one or more genes by artificial means.

No animals yet but plenty of crops

Biosafety Protocol of 2000: exporters must identify GMOs present in their bulk food shipments and the importing countries can decide if there is a health or environmental risk.

There is the fear that crops carrying genes from other species are a health concern and could do ecological harm.

So a transgenic plant could transfer the new genes to a closely related species that was never intended to be modified. So if a weeds we want to control picked up a gene from a modified relative that was herbicide resistant, we would have a hard time controlling the weed.

No scientific evidence that genetically modified crops to resist pests pose a risk.

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Figure 20.x2 Injecting DNA

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The Technique of Restriction Fragment Length Polymorphisms

1) What is a Riff-Lips (RFLP)? Mutations within a genome, coding or noncoding regions, can affect where restriction enzymes can cut. The addition or deletion of a restriction site will then affect the size and number of fragments.

2) This can occur with in a gene (coding region) or a region flanking the gene (noncoding region)

3) This will produce different “restriction maps” between individuals on the same chromosome.

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4) The technique

-obtain DNA

-cut with restriction enzymes

-electrophorese

-separation of size of fragments based on size

-stain with ethidium bromide

-photograph and analyze

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What has this technique contributed to genetics?

1) Genetic markers in families

2) Marker for diseases: sickle cell has only one amino acid change because an AT base pair is changed to a TA base pair. This changes the codon and valine is inserted instead of glutamic acid in the Beta-globin polypeptide of hemoglobin. This single base pair substitution creates a restriction site thus creating a different RFLP pattern between affected and normal individuals.

3) Phenylketouria (PKU) is diagnosed by a RFLP caused in a flanking region of a gene responsible for making the enzyme phenylalanine hydroxylase. Tested by amnio- or chorionic villus sampling in the fetus.

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4) DNA fingerprinting

5) Mapping of DNA can be done by RFLP: if two RFLPs are inherited frequently or if a RFLP marker and an allele are frequently inherited, this can be used as a measure closeness of the two segments of DNA (the two loci).

6) Evolutionary relationships between plants / animals.

7) Find mutations

8) Sequences of DNA can be determined even though you do not know its function.