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9/24/2018

BIO374 Modern Biotechnology


Recombinant
DNA and Genomics

<Important Issues>

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
• 1970s: Gene cloning

– Clone

• Made possible by the discovery of:

– Restriction Enzymes

– Plasmid DNA Vectors


© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• Restriction Enzymes
–bacteria
–phosphodiester bond
–Bind to, recognize, and cut DNA
–restriction site
• same forward and backward
–4 or 6 bp cutters
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Test your understanding!

• Why don't restriction enzymes digest bacteria DNA?


© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Test your understanding!

• Why don't restriction enzymes digest bacteria DNA? Methyl


groups block restriction enzymes from digestion
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
• Restriction enzymes

a."sticky" or "cohesive" ends

b."blunt" ends

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• Plasmid DNA
• extrachromosomal DNA
• approximately 1 to 4 kb
• replicate independently of
chromosome
• vectors

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
• Creating recombinant DNA

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• Transformation of Bacterial Cells

–very inefficient process

–inserting foreign DNA into bacteria

–electroporation
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
• Selection of recombinant bacteria after transformation

-Antibiotic selection

-Blue-white selection

artificial lactose

lacZ is present = blue colony

non-functional lacZ = white colony


© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Test your understanding!


• Assume you used a plasmid that contains the lacZ
gene in the restriction enzyme site. The plasmid has
an antibiotic resistance gene. Following
transformation, you grow up the cells on an agar
plate containing the antibiotic. Here are your results.
• Work in groups of two and discuss which colonies
have the inserted gene.

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Test your understanding!


• Assume you used a plasmid that contains the lacz
gene in the restriction enzyme site. The plasmid has
an antibiotic resistance gene. Following
transformation, you grow up the cells on an agar
plate containing the antibiotic. Here are your results.
• Work in groups of two and discuss which colonies
have the inserted gene. 1, 3 and 6

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
• human gene cloning

• First human protein expressed via


recombinant techniques

insulin

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• Types of Vectors
– Bacterial plasmid vectors
inserts that are smaller than 7 kb
– Bacteriophage vectors
– Cosmid vectors
– Expression vectors
– Bacterial Artificial Chromosomes (BAC)
– Yeast Artificial Chromosomes (YAC)
– Ti vectors

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
• Bacteriophage vectors

– advantage: clone up to 25 kb

–infect E coli cells

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• Cosmid vectors:

• advantage: clone fragments


between 20–40 kb

• infect E coli cells

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
• Expression vectors
– Bacteria expression vector:
• prokaryotic promoter site
• Bacterial RNA POL can bind to promoter
• Problems?
-bacteria ribosomes cannot translate
eukaryote sequence
-protein is not folded correctly

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Test your understanding!


• Assume you want to make lots of human insulin
using a bacteria expression vector.
• Work in groups of two and discuss why using
human insulin genomic DNA for this cloning
project would not be advantageous.

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Test your understanding!


• Assume you want to make lots of human insulin
using a bacteria expression vector.
• Work in groups of two and discuss why using
human insulin genomic DNA for this cloning
project would not be advantageous.
No splicing in bacteria -> contain introns ->
wrong protein

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• Bacteria artificial chromosomes
(BAC)
–Large low copy plasmids

–sizes of DNA inserts ranging from


100–300 kb

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
• Yeast artificial chromosomes (YAC)

–DNA inserts from 200 kb to 2


megabases

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• Ti vector
–isolated from the bacterium
–bacteria infects plant cells
–T DNA from the Ti plasmid inserts
into the host chromosome
–T DNA codes for auxin
–use Ti vectors to deliver genes to
plants
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

How do you identify and clone a gene


of interest?

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• Creating DNA Libraries

– Collections of cloned DNA fragments

Two Types of Libraries

– Genomic DNA libraries

– Complementary DNA libraries (cDNA libraries)


© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
–Disadvantages of genomic
libraries
•Introns are cloned in addition to
exons
•very large genome
•Time consuming!
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• cDNA Libraries
– Advantage over genomic libraries
• Collection of actively expressed genes
• Introns are NOT cloned
• isolate genes that are primarily expressed
only under certain conditions
– Disadvantage
• source tissue with an abundant amount of
mRNA
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
• Colony hybridization

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• Polymerase Chain Reaction

–mid-1980s by Kary Mullis


–making copies, or amplifying
–Process
• thermocycler

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Test your understanding!


• Advantage of PCR:
• Can amplify millions of copies of target DNA from
small amount of starting material in short period of
time
• To calculate the number of copies of target DNA
starting with 1 molecule of DNA use this equation 2N
in which N represents number of PCR cycles
• Assume you want to do 22 PCR cycles to amplify your
DNA insert, how many copies of DNA will you have
at the end of your PCR?

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Test your understanding!


• Advantage of PCR:
• Can amplify millions of copies of target DNA from
small amount of starting material in short period of
time
• To calculate the number of copies of target DNA
starting with 1 molecule of DNA use this equation 2N
in which N represents number of PCR cycles
• Assume you want to do 22 PCR cycles to amplify your
DNA insert, how many copies of DNA will you have
at the end of your PCR? 222 = 4,194,304

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Test your understanding!


• The type of DNA polymerase used is
very important
–Taq DNA polymerase – isolated from
a species known as Thermus
aquaticus that thrives in hot springs
–Why can't you use DNA Pol isolated
from bacteria that live at 37 C?

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Test your understanding!


• The type of DNA polymerase used is
very important
–Taq DNA polymerase – isolated from
a species known as Thermus
aquaticus that thrives in hot springs
–Why can't you use DNA Pol isolated
from bacteria that live at 37 C?
Enzyme will be denatured
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts

• Cloning PCR Products


–Is rapid and effective

–Disadvantage
• Need to know something about the
DNA sequence that flanks the gene
of interest to design primers
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Any laboratory techniques and


applications of recombinant DNA
technology?

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Test your understanding!


• Agarose Gel Electrophoresis:
-size
-a semisolid gel containing small pores
-(gel % range from 0.5 to 2%)
-High % gel (2%) allows to resolve smaller size fragments
-Low % (0.5%) resolves larger size fragments

• What size fragments would be resolved using a 1% gel?

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Test your understanding!


• Agarose Gel Electrophoresis:
-size
-a semisolid gel containing small pores
-(gel % range from 0.5 to 2%)
-High % gel (2%) allows to resolve smaller size fragments
-Low % (0.5%) resolves larger size fragments

• What size fragments would be resolved using a 1% gel?


Medium sized fragments

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
• Agarose Gel Electrophoresis

–buffer solution
–wells at the top of the gel
–Electric current is applied through
electrodes at opposite ends of the
gel
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• Agarose Gel Electrophoresis

-Migration distance

–Tracking dye
–DNA staining dyes

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Test your understanding!

• Here is a result in which you ran your DNA fragments on a 1% gel. What
is the approximate size of band B? Why did it not migrate super fast
through the gel?

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Test your understanding!

• Here is a result in which you ran your DNA fragments on a 1% gel. What
is the approximate size of band B? Why did it not migrate super fast
through the gel? B is 1250bp; B is not the smallest fragment

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
• DNA Sequencing

–Sanger
–Roche 454
–ABI SOLID
–Nanotechnology

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• Fluorescence in situ
hybridization (FISH)

-Chromosome Location

-Copy Number

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Test your understanding!

What does fluorescence on more


than one chromosome indicate?

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Test your understanding!

What does fluorescence on more


than one chromosome indicate?

Multiple copies of the gene

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
–Southern blotting

–determine gene copy number;


gene mapping; gene mutation
detection; PCR product
confirmation; DNA fingerprinting

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
Microarrays

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts

Gene microarrays

–2 different colored dyes

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
RNA
interference
(RNAi)

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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What are genomics and


bioinformatics?

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts

• Genomics – cloning, sequencing, and


analyzing entire genomes

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• Bioinformatics: Merging molecular
biology with computer technology

• Application
–Databases

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
–GenBank

• accession number

• Maintained by the National


Center for Biotechnology
Information (NCBI)
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• Basic Local Alignment Search
Tool (BLAST)

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Key Concepts
• The Human Genome Project
–Started in 1990 by the U.S. Department
of Energy
–International collaborative effort
–China, France, Germany, Great Britain,
Japan, and the United States
–Competitor: Celera Genomics

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

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Key Concepts
• The Human Genome Project
– April 14, 2003: map of the human genome was completed
– Consists of 20,000 protein-coding genes
– Map was complete with virtually all bases identified and
placed in their order and potential genes assigned to
chromosomes

• Why are there only 20,000 genes coding for proteins when it
was predicted that there would be 100,000 genes?

© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture

Next Class:
BIO374 Modern Biotechnology

Proteins as Products

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