PCR Review

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

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

Original Sample DNA Large quantities of oligonucleotide DNA primers

(Anneal)– 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

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

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.– 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?

Sanger Method utilized in Cycle Sequencing Many copies of DNA are made in a Thermocycler Added to a reaction mixture:

– 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.

– Dideoxynucleotides - http://www.dnalc.org/ddnalc/resources/sangerseq.html

Dideoxynucleotide (didNTP)

Dideoxynucleotide – didNTP

Terminates replication Dideoxynucleotides can substitute for normal

deoxynucleotides during replication– 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

Effort to Map the entire human genome– 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 Small number of Human Genes

– 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.

– Through comparative analysis genes of other species help scientists interpret human data.

Challenges of Human Genome

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

Includes Introns and non-coding DNA located between genes

Much of the DNA between genes consists of repetitive DNA

– 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

Read Human Genome Project (HGP) Articles In notebook

– 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

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.

– Protein products of Recombinant DNA Technology: Human Insulin Hepatitis B vaccine Erythropoietin (EPO)

LosatSoy

What was this article about? What health risk were they trying to address? What are some other potentially beneficial

GM foods.

ACME FOODS INC.

Start Lab.

ACME FOODS INC.

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? – Would you want GM food labels ?

Genetically Modified (GM) Foods

Human Population is growing exponentially– 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

Ti Plasmid is utilized as a vector– 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:

– 74 % of American soybean crop– 32% of the corn crop– 71% of the cotton crop

GM Benefits

Herbicide resistance Microbe resistance Pest resistance Improved Nutritional Content

– Golden Rice

LosatSoy Your Product?

Could GM Organisms harm human health or the environment?

Transferring of New Types of Allergens in GM foods

Transgenic plants may pass their genes to close relatives in nearby wild areas. – Could produce “Superweeds.”

Case Study – The monarch butterfly

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.

– 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

“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

What did Leroy Hood mean? What are the problems with being able to

modify DNA? Who should have control over genetic

information?