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PCR Review
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Collect Lab Write-ups
What is PCR?
What are the 3 steps essential for
amplification?
What are the essential reagents/ingredients?
PCR Amplification:
www.hhmi.org/biointeractive
3 Essential Steps
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Step 1: Melt – Separate the two DNA chains in the
double helix by heating the vial containing the PCR
reaction mixture to 95°C for 30 seconds.
Step 2: Anneal – The primers cannot bind to the
DNA strands at such a high temperature, so the vial
is cooled to 60°C. At this temperature, the primers
bind (anneal) to the single-stranded DNA.
Step 3: Extend – is to allow the DNA polymerase
to extend the copy DNA strand by raising the
temperature to 70°C for 45 seconds.
Essential Reagents
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Original Sample DNA
Large quantities of oligonucleotide DNA primers
(Anneal)
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Primers are small pieces of DNA that bind to specific
sequences
Heat-stable DNA polymerase that extends the copy
DNA strand. (Extend)
Large quantities of the four nucleotides adenine,
cytosine, guanine, and thymine (Extend)
PCR
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Amplifies a DNA when only small sample of
DNA
Resulting multiple copies of DNA can be
manipulated in various lab applications –
DNA Fingerprinting, DNA Sequencing
DNA Sequencing
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Virtually all the information required for growth and
development of an organism is encoded in the DNA of its
genome.
DNA restriction enzymes can break up nucleotides into
restriction fragments a few hundred bases long.
PCR can help amplify DNA and make multiple copies
This has led to the need for development of methods to
determine the exact sequence of stretches of DNA
Currently we are only able to accurately sequence around 600
bases accurately – great start, but only a start.
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Human genome is over 3 Billion bases long, arranged on 23
chromosomes.
How do we find out what is the exact
sequence of bases in a DNA fragment?
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Sanger Method utilized in Cycle Sequencing
Many copies of DNA are made in a Thermocycler
Added to a reaction mixture:
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that contains normal deoxynucleotides (A, C, G, and T)
special dideoxynucleotides (A*, C*, G*, and T*) that are tagged with
fluorescent markers. The fluorescent markers differ for each base, and
are designed to fluoresce with different colors (G* is yellow, T* is
green, for example)
Utilizing the Sanger method you terminate the replication process
at random places with dideoxynucleotides so the copies are all
partial sequences with different lengths.
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Dideoxynucleotides http://www.dnalc.org/ddnalc/resources/sangerseq.html
Dideoxynucleotide (didNTP)
Dideoxynucleotide – didNTP
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Terminates replication
Dideoxynucleotides can substitute for normal
deoxynucleotides during replication
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when such a substitution occurs by chance, that
chain can no longer be extended, terminating that
DNA strand.
Dideoxynucleotides are thus called terminators.
Gel Electrophoresis
Automated sequencing gel
Computer Sequencing Output
Adding all 4 deoxynucleotides (dNTP)
into the picture
(DID NOT GO OVER IN CLASS – OMIT)
Human Genome Project
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Effort to Map the entire human genome
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Small number of Human Genes
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National Institute of Health and National Science Foundation have
funded the creation of libraries of gene maps.
Researchers use restriction enzymes to break the DNA into a
number of identifiable fragments
30-40,000 genes. Only 2 or 3 times the number found in the fruit
fly and nematode worm.
Many genes of disparate organisms are turning out to be very
similar.
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Through comparative analysis genes of other species help
scientists interpret human data.
Challenges of Human Genome
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Finding exact DNA sequences is time intensive
The Genome is enormous – Approximately 3 Billion
nucleotide pairs of DNA
Only 35,000 genes have been identified as protein
coding or genes for tRNA and rRNA.
About 97% of the total human DNA is noncoding
DNA – “Junk DNA”
Junk DNA does not code for proteins – we do not
yet understand its functions.
Junk DNA
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Includes Introns and non-coding DNA located
between genes
Much of the DNA between genes consists of
repetitive DNA
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Such repetitions found at the centromeres and ends of
chromosomes, suggest this DNA plays a role in
chromosome structure
Telomeres – are repetitive DNA found at the ends of
chromosomes may have a protective function.
Human Genome Homework
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Read Human Genome Project (HGP) Articles
In notebook
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List 5 benefits of the HGP
List 3 challenges of the HGP
Identify 2 ethical issues with genomics research.
Potential Benefits of Human Genome
Project
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Insight into the mysteries of embryonic development and
evolution
Will aid in the diagnosis, treatment, and possible
prevention of common ailments like heart disease,
allergies, diabetes, etc.
Ability to identify hundreds of disease-associated
genes—Alzheimer’s and cancer.
By transferring a gene of interest into a bacterial vector
useful proteins can be made.
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Protein products of Recombinant DNA Technology:
 Human Insulin
 Hepatitis B vaccine
 Erythropoietin (EPO)
LosatSoy
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What was this article about?
What health risk were they trying to address?
What are some other potentially beneficial
GM foods.
ACME FOODS INC.
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Start Lab.
ACME FOODS INC.
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Which product would you produce? Why did you
choose that product?
What are the concerns or issues that this product
might bring up? Who are the people involved in this
issue?
Who will do the testing for your product? What type
of testing needs to be done?
Will you label your food product? Why or Why not?
How will you promote your product to consumers?
As a consumer, how would you make a decision
about whether to purchase this product?
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Would you want GM food labels ?
Genetically Modified (GM) Foods
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Human Population is growing exponentially
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Currently about 6.5 Billion people on the planet.
In the next 50 years we will be over 9 Billion.
Concern will be how will we feed the growing
human population?
GM Organisms – acquire one or more
genes by artificial means rather than by
traditional breeding methods.
GM Plants
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Ti Plasmid is utilized as a vector
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Gene of interest is inserted to form a recombinant plasmid
and introduced into plant cells
If the newly acquired gene is from another species,
the recombinant organism is called a transgenic
organism.
US Department of Agriculture estimates of
genetically modified crops:
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74 % of American soybean crop
32% of the corn crop
71% of the cotton crop
GM Benefits
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Herbicide resistance
Microbe resistance
Pest resistance
Improved Nutritional Content
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Golden Rice
LosatSoy
Your Product?
Could GM Organisms harm human
health or the environment?
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Transferring of New Types of Allergens in GM
foods
Transgenic plants may pass their genes to
close relatives in nearby wild areas.
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Could produce “Superweeds.”
Case Study – The monarch butterfly
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Monarchs migration paths can span up to 3,000
miles long.
They travel through forests and over fields, including
more and more acres growing Bt corn.
Bt Corn has been genetically modified to produce a
pesticide.
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Pesticide is found in all parts of the plant – including pollen
Pollen can drift off onto leaves of nearby milkweed plants.
Milkweed plants are the favorite food for Monarch
caterpillars.
Some researchers claimed the Bt corn stunted or
killed the monarch caterpillars
Leroy Hood
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“What science does is give society
opportunities. What we have to do is look at
these opportunities and then set up the
constraints and the rules that will allow
society to benefit in appropriate ways.”
Issues
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What did Leroy Hood mean?
What are the problems with being able to
modify DNA?
Who should have control over genetic
information?