Outline the use of polymerase chain reaction (PCR) to copy ... · 4.4: Genetic Engineering and Biotechnology • 1. DNA fragments are placed in a well in a plate of gel (material

4.4: Genetic Engineering and Biotechnology

★Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA.

★State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size.

★State that gel electrophoresis of DNA is used in DNA profiling.


★Describe the application of DNA profiling to determine paternity and also in forensic investigations.

★Analyze DNA profiles to draw conclusions about paternity or forensic investigations.


★Outline 3 outcomes of the sequencing of the complete human genome.

★State that, when genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal.


★Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast or other cell), restriction enzymes (endonucleases) and DNA ligase.

★State 2 examples of the current uses of genetically modified crops or animals.

★Discuss the potential benefits and possible harmful effects of one example of genetic modification.


★Define clone.

★Outline a technique for cloning using differentiated animal cells.

★Discuss the ethical issues of therapeutic cloning in humans.

• Genetic engineering and biotechnology have opened up new opportunities in forensic science (you know this as CSI), agriculture, medicine and food technology.

• We are going to discuss in depth how science has allowed people to change the genetic identity of organisms - GMO's and Cloning!


• DNA Profiling: matching the DNA from a sample to a known individual.

• At a crime scene, forensic scientists check for fingerprints, collect samples of hair, skin, blood, semen, and anything else that can be used to identify the suspect.

• DNA profiling, can not only establish if a person is guilty or innocent, but can be used to determine paternity because DNA is unique to that individual

• Making a DNA profile requires 2 parts:

• 1. Polymerase Chain Reaction (PCR)

• 2. Gel Electrophoresis.


• 1. Polymerase Chain Reaction (PCR): makes millions of copies of tiny amounts of DNA so there is enough for a DNA profile.

• Sometimes, at a crime scene or when a body is found after a very long time, only a small amount of DNA can be collected.

• Making a DNA profile on someone only works with a significant amount of DNA.

• PCR uses the segment of DNA that needs to be replicated, as a template, setting in motion a chain reaction in which the DNA template is exponentially amplified.


• Making a lot of DNA is done at a high temperature using the enzyme DNA POLYMERASE.

• PCR targets a specific DNA sequence and allows the enzyme DNA polymerase to assemble a new DNA strand from DNA building blocks (nucleotides)

• Temperature both heats up and cools down the strand - allowing strands to separate and re form as new DNA is made at a rapid pace.

• Sum up PCR: to copy and amplify (enlarge) small quantities of DNA



PCR!

• 2. Gel Electrophoresis: method used to separate DNA fragments based on their size and electric charge.

• After you use PCR to amplify a small amount of DNA you found at a crime scene, how can you tell if the suspect is guilty who is the dad of a child?

• Run the DNA sample through a Gel Electrophoresis!



• Any DNA sample usually contains molecules that are too long to process to make a DNA profile.

• Restriction Enzymes are used to cut DNA into fragments at very precise points in the sequence.

• Because everyone has a different sequence, an individual is going to have unique DNA fragments that are cut.

• Thus, each person will have DNA fragments of different sizes.

4.4: Genetic Engineering and Biotechnology • 1. DNA fragments are placed in a well in a plate of gel (material like jell-o)

• 2. An electric field is applied.

• 3. Each DNA fragment has a small negative charge so they will move through the electric field in the gel to the positive charge, all the way at the end of the gel.

• 4. The distance a fragment moves depends on its size.

• Smaller fragment = farther distance traveled in gel

• Larger fragment = short distance traveled, stay by the well

• 5. After fragments have been separated, what is produced is a unique pattern of bands called a DNA profile


4.4: Genetic Engineering and Biotechnology • In each picture, circle the guilty suspect or

real father!

4.4: Genetic Engineering and Biotechnology • In each picture, circle the guilty suspect or

real father!

Bellringer 1

•  Based on this evidence, which suspect is likely guilty?!

Bellringer 2 •  Based on this evidence, which suspect is likely

guilty?!

Bellringer 3 •  Based on this evidence, which Allele (Out of A, B,

and C) ends up yielding the smallest pieces after being treated with restriction enzymes? (Hint: Keep in mind DNA has a negative charge)!


• The Human Genome Project (HGP):

• In 1990, an international venture (HGP) set out to sequence the complete human genome.

• 20,000-25,000 genes, location of all these genes on the human chromosomes and the base sequence of the DNA that makes them up.

• A genome is a catalog of all the bases (A/T/G/C) our chromosomes contain.

• The human genome can be thought of as a map to show the locus of any gene on any one of the 46 chromosomes.


• The $3 billion project was given 15 years, but it was completed in 13 - in 2003.

• Since then, scientists have completed the genomes of other organisms, such as E. coli, fruit fly, and field mouse.


• Advantages/outcomes of the HGP:

• 1. Easier identification of human diseases

• As you have seen, some diseases are sex-linked, so with those diseases it is easy to tell which chromosome the gene responsible for the disease is found on - almost always on the X chromosome.

• In traits/diseases with no sex linkage, a little bit trickier.

• Now that the HGP completed, doctors can look on the genome library to find out exactly where to look if they think one of their patients might have a disease-carrying allele.


• 2. Production of new drugs

• Scientists can find beneficial proteins which are produced naturally in healthy people.

• Find out which gene controls the production of those proteins.

• Copy that gene and use it as instructions to make that protein in a lab.

• Scientists are working on gene therapy: drugs that work to repair or replace a faulty gene - essentially drugs tailored to a specific individual.


• 3. Insights into the origins, evolution and migrations of humans.

• By comparing the genetic makeup of populations around the world, countless details could be revealed about ancestries and how humans have migrated and mixed their genes with other populations over time.

• Without knowing it, you are carrying around in each one of your cells a library of information about your past.


• Gene Technology:

• Selective plant and animal breeding has been carried out by humans for thousands of years as people tried to develop cattle that produced high milk yields or crops with better resistance to disease.

• In these cases, animals or plants of the same species were chosen for breeding because of their particular characteristics (think Gregor Mendel only bred pea plants with other pea plants).

• Over many generations of selection, these desired characteristics increased in frequency in the population.


• Today, with access to incredible technology, transferring genes isn't just limited to one species.

• Gene technology allows us to transfer DNA from one species to another completely different species in just a single generation.

• For example, bacteria genes have been transferred to plants, human genes transferred to bacteria, and spider genes transferred to a goat.

4.4: Genetic Engineering and Biotechnology • Gene Transfer:

• Organisms that have had genes transferred to them are called genetically modified organisms (GMO)

• It is the technique of taking a gene out of one organisms (donor) and placing it into another organism (host).

• It is possible to put one species' genes into another's genetic makeup because DNA is universal!


• Remember all living organisms have the same bases (A, T, G, C) which code for proteins.

• The codons they form always code for the same amino acids, so transferred DNA codes for the same polypeptide chain in the host organism as it did in the donor organism.

Arctic Fish!Gene for

resistance to cold!

Genetically engineered

tomato is more resistant to cold!


• Gene transfer is found in Bt-corn, which has been genetically engineered to produce toxins that kill the bugs that attack it.

• The gene comes from a soil bacterium, Bacillus thuringiensis, thus Bt, which has the ability to produce a protein that is fatal to the larvae of certain crop-eating pests.

Bacteria! Gene for pest-killing toxin!

Genetically engineered corn

is resistant to pests!


• Gene transfer is possible because the genetic code is universal.

• No matter what the species, amino acids translated to polypeptides, then proteins, stays the same.

• Usually, in gene transfer, only one gene at a time is transferred to another species.

• EX: our gene for blood-clotting has been transferred to sheep which produce this gene in their milk.

• Humans drink this milk who have blood clotting problems.


• One of the first important uses of gene transfer was to produce insulin for diabetic patients who do not produce insulin properly.

• Many years ago, insulin was obtained from cow or pic pancreases but the process was difficult and the insulin was likely to be contaminated.

• Today, diabetics inject themselves with human insulin that has been made by modified E. coli bacteria.

• This has enormous benefits, and no obvious harmful effects - but other gene transfers are much more controversial.

• So how did scientists do that? ......


• Steps to Gene Transfer:

• 1. Plasmid (circular DNA molecule) is removed from the host cell.

• 2. Plasmid is cut open using a restriction enzyme called endonuclease

• 3. The gene to be copied is placed inside the open plasmid. This is called gene splicing.

4.4: Genetic Engineering and Biotechnology • 4. The gene is pasted into the plasmid using DNA ligase.

The plasmid is now called a recombinant plasmid and it can be used as a vector - a tool for introducing a new gene into an organism's genetic makeup.

• 5. Finally, the vector is placed inside the host bacterium and the bacterium is given its ideal conditions to grow and proliferate (multiply). This is done by putting the bacterium into a bioreactor, a vat of nutritious liquid kept at a warm temperature.




• Genetically Modified Organisms (GMOs):

• A GMO is one that has had an artificial genetic change using the techniques of genetic engineering such as gene transfer or recombinant DNA.

• FIRST EXAMPLE of GMO: Food Production

• Maize crops are often seriously damaged by corn borer insects.

• A gene from a bacterium (Bacillus thuringiensis, or Bt) can be transferred to maize.

• Then this gene transfer allows corn to produce the Bt toxin that kills the insect's larvae.


• SECOND EXAMPLE of GMO: Production for Medical Treatment

• Some people's blood does not clot because they lack a protein called factor IX.

• Least expensive way for these people to get the protein they need is by producing large amounts of factor IX by using sheep.

• Sheep are transferred with the gene that makes protein/factor IX, and that gene is inserted in part of the DNA that codes for milk production.

• This milk now contains factor IX for human consumption.

Gene for human protein factor IX!

GM sheep which synthesized factor IX!

Milk containing factor IX!


GMO BENEFITS!

GM crops will help farmers improve food production!

Much faster than selective breeding (what has been done for thousands of

years)!

GM crops which produce their own pest-control substances will be

beneficial to the environment because fewer chemical pesticides are needed!

Multinational companies who make GM plants claim they will enable

farmers in developing nations to help reduce hunger by using pest-resistant crops or GM plants that require less

water!

Using GMOs to produce rare proteins for medications or vaccines could be, in

the long run, less costly and produce less pollution than making proteins in

labs.!

Farmers can be more in control of what crops or livestock they produce. There

is always some randomness in breeding, so GM makes the process less

of a gamble.!


GMO CONCERNS!

Increasing number of GMOs may lead to a decrease in biodiversity!

Large portions of human food supply can be property of a small number of

corporations!

Risks for allergies - someone may not be allergic to natural tomatoes but is

allergic to GM tomatoes, they will need to know difference. But there is no

difference in the outward appearance and labeling isn't mandatory.!

Bt-crops which produce toxins to kill insects could be harmful to humans because, unlike chemical pesticides which are only applied to the outer

surface, the toxins are found throughout the plant.!

Danger that genes could cross species. It has been proven possible in labs, so there is a possibility in nature too. No one knows the consequences of genes

crossing species.!

What are long term effects? Efforts to keep GM plants contained in an area

have failed and pollen from GM crops has escaped to nearby fields. Genes could be integrated into wild species giving them an unnatural advantage.!

Topic 4: Genetics

• 30. Which disease is an example of sex-linked (X-linked) inheritance?

• A. AIDS

• B. Down syndrome

• C. Sickle-cell anemia

• D. Hemophilia

• Answer: D

Topic 4: Genetics

• 31. Which process is used in polymerase chain reaction (PCR)?

• A. Transcription

• B. Translation

• C. Replication

• D. Mutation

• Answer: C

Topic 4: Genetics

• 32. How can fragments of DNA be separated?

• A. Using polymerase chain reaction (PCR)

• B. Using gel electrophoresis

• C. Using gene transfer

• D. Using gene cloning

• Answer: B

Topic 4: Genetics • 33. What could be achieved by DNA profiling using gel

electrophoresis?

• A. The chromosome number of an organism could be counted.

• B. It could be shown that human tissue found at the site of a crime did not come from a person suspected of having committed the crime.

• C. A karyotype could be produced.

• D. Extinct species of living organisms could be brought back to life.

• Answer: B

Topic 4: Genetics • 34. Why can DNA profiling be used to determine paternity?

• A. Genes of children are exactly the same as their father's

• B. Half the genes of children are the same as their father's

• C. The father passes on all of his genes to each of his children

• D. The father passes on a fraction of his genes equal to the number of his children.

• Answer: B

Topic 4: Genetics • 35. The diagram below represents the results

obtained in a DNA profile from a crime scene. Suspect 2 is most likely to be the criminal because the band pattern concides with that of the crime scene sample. What do these bands represent?

• A. DNA fragments

• B. Genes

• C. Chromosomes

• D. Chromatids

• Answer: A

Topic 4: Genetics • 36. What conclusion can be made

from the following evidence from an analysis of DNA fragments?

• A. Both children are related to both parents

• B. Child I is related to the man but child II is not

• C. Both children are unrelated to either of the parents

• D. Child II is related to the man but child I is not

• Answer: A

Topic 4: Genetics

• 37. A small amount of a suspect's DNA is obtained from a crime scene. What techniques would be used to carry out DNA profiling?

• A. Gel electrophoresis and paternity testing

• B. Paternity testing and the polymerase chain reaction (PCR)

• C. Polymerase chain reaction (PCR) and gel electrophoresis

• D. Test crossing and pedigree analysis

• Answer: C

Topic 4: Genetics • 38. The Human Genome Project allowed the first

accurate estimates of the number of different genes in the human genome. What was a typical estimate, based on the results of the Human Genome Project?

• A. 46

• B. 64

• C. 25,000

• D. 1,000,000

• Answer: C

Topic 4: Genetics

• 39. How are plasmids used in technology?

• A. For respiration in prokaryotes

• B. For photosynthesis in eukaryotes

• C. For protein synthesis in prokaryotes and eukaryotes

• D. For gene transfer

• Answer: D

Topic 4: Genetics

• 40. What type of enzyme could be used to cut a DNA molecule as indicated by the dotted line on the diagram below?

• A. DNA ligase

• B. DNA polymerase

• C. Helicase

• D. Restriction enzyme

• Answer: D

Topic 4: Genetics

• 41. Which enzymes are needed to incorporate genes into plasmids to create recombinant plasmids?

• A. DNA polymerase and ligase

• B. DNA polymerase and restriction enzymes

• C. Restriction enzymes and ligase

• D. Helicase and restriction enzymes

• Answer: C

Topic 4: Genetics • 42. Which enzymes are needed to produce

recombinant plasmids to be used in gene transfer?

• A. DNA polymerase and DNA ligase

• B. DNA polymerase and restriction enzyme (endonuclease)

• C. Transcriptase and RNA polymerase

• D. Restriction enzyme (endonuclease) and DNA ligase

• Answer: D

• 43. Which processes involved in cloning an animal are indicated by the letters X and Y?

• Answer: A

X! Y!

A! differentiated cell removed from animal!

nucleus removed from unfertilized egg cell!

B! sex cell removed from animal!

nucleus removed from differentiated animal cell!

C! sex cell removed from animal!

nucleus removed from unfertilized egg cell!

D! differentiated cell removed from animal!

nucleus removed from differentiated animal cell!

Documents

Outline the use of polymerase chain reaction (PCR) to copy ... · 4.4: Genetic Engineering and Biotechnology • 1. DNA fragments are placed in a well in a plate of gel (material