PSU BI 341 CHAPTER 10

GENOMICS, PROTEOMICS, AND GENETIC

ENGINEERING
Kim H. Brown, PhD
 Genetic Engineering
Recombinant DNA: created by joining together pieces of DNA
from different sources.
Recombinant DNA Technology is used to copy or clone DNA,
which allowed scientists to isolate and study specific DNA
sequences.
• Two important tools are used to construct and amplify DNA
molecules.
• DNA-cutting enzymes: restriction enzymes
• DNA cloning vectors

Cloning a gene:
http://highered.mheducation.com/sit
es/0072556781/student_view0/chap
ter14/animation_quiz_1.html
 Objectives
1. Recombinant DNA technology
a. Restriction enzymes
b. Ligation
c. Cloning into vectors
d. Identifying Genes of Interest: cDNA libraries
e. Detection of bacteria transformed with recombinant
cDNA
2. Applications of genetic engineering
3. Analysis of genomic sequences: Bioinformatics &
functional genomics
a. Reverse genetics
b. Transformation genetics
c. Microarrays
Restriction enzymes
• A restriction enzyme recognizes and binds to DNA at a
specific nucleotide sequence (restriction site) and cleaves
the DNA to produce restriction fragments Restriction sites are
present randomly in the genome.
• These cuts can
produce fragments with
single overhanging
ends called cohesive
ends (sticky ends),
while cuts on both
ends in the same
nucleotide produce
blunt-end fragments.
 Restriction enzymes
• Most recognition
sequences are
palindromic (the
nucleotide sequence
reads the same on both
strands in the
5–3 direction)
• Each restriction
enzyme cuts the DNA
in a characteristic
cleavage.
• DNA ligase seal the
joints
 Restriction Enzymes
• Sticky ends formed by restriction enzymes permit circularization
of the DNA restriction fragment by complementary base pairing
• Joining a donor DNA fragment of interest to a vector is the
basis of the recombinant DNA technology
 DNA Cloning
In genetic engineering, DNA fragment of interest is joined to a
vector, a DNA molecule that is able to replicate inside a cell
and contains sequences that confer antibiotic resistance (or
some other detectable phenotype)

Construction of a Plasmid Vector:

http://highered.mheducation.com/sites/0072556781/student_view0/chapter14/animation_quiz_2.html
 DNA Cloning
• The simplest types of
vectors are plasmids
whose DNA is
double-stranded and
circular

• When the DNA

fragment has been
joined to the vector,
the recombinant
molecule is
introduced into a cell
by means of DNA
transformation
 DNA Cloning
• When a transformant containing the recombinant molecule
has been isolated, the DNA fragment linked to the vector is
said to be cloned
 DNA Cloning: Vectors
The most useful vectors have four properties:
1. The vector DNA can be introduced into a host cell
relatively easily
2. The vector contains a replication origin and so can
replicate inside the host cell
3. The vector contains a multiple cloning site (MCS), or
polylinker, with unique cleavage sites for many different
restriction enzymes that enables many types of
restriction fragments to be inserted
4. Cells containing the vector can usually be selected by a
straightforward assay, most conveniently by allowing
growth of the host cell on a solid selective medium
 Cloning Vectors
Three types of vectors commonly
used for cloning into E. coli:
 Plasmids are most
convenient for cloning
relatively small DNA
fragments (5-10 kb)
 Fragments (from 12-20 kb)
can be cloned with
bacteriophage 
 Still larger DNA fragments
(40-45 kb) can be inserted
into cosmid vectors. These
vectors can exist as
plasmids, but they also can
be packaged into mature
phages.
 Cloning Vectors: BACs
• Among the most widely used are bacterial artificial
chromosomes (BACs)
• The BAC vector is based on the F factor of E. coli and includes
genes for replication
 cDNA Cloning: selecting the gene of interest for
insertion into a vector
Complementary DNA (cDNA) libraries:
contain complementary DNA copies
(cDNA) made from the mRNAs to isolate
expressed genes.
1. Reverse transcriptase synthesizes
a complementary DNA (cDNA) from
an mRNA template.
2. Reverse transcriptase requires a
primer. The primer can be a poly-T
oligonucleotide which targets the
poly-A tail
3. The resulting full-length cDNA
contains an uninterrupted by introns
coding sequence for the protein of
interest
 cDNA Cloning
• The single strand forms at the 3’ end “hairpin” that serves as
a primer for second-strand synthesis.
• Conversion into a conventional double-stranded DNA
molecule is achieved by cleavage of the hairpin by a
nuclease

cDNA:
http://highered.mhed
ucation.com/sites/007
2556781/student_vie
w0/chapter14/animati
on_quiz_3.html
 Combining techniques: Making a biological
machine to mass produce clones of cDNA library
1. Generate recombinant plasmids of vectors containing cDNA
2. Transform recombinant plasmids into bacterial cells
3. Isolate bacteria that contain the plasmid from a mixture of
plasmid-free and plasmid-containing cells.
• Detecting transformed bacteria: Use a plasmid that has an
antibiotic-resistance marker and to grow the transformed
bacteria on a medium that contains the antibiotic: Only cells
that contain plasmid can form a colony.
 Detection of Recombinant Plasmids
A the lacZ gene is interrupted by a
fragment of DNA inserted into the MCS
1. The recombinant plasmid is Lacz-
2. Nonrecombinant plasmids are Lacz+
3. NOTE: Lacz gene + X-gal = subtrate +
enzyme = blue colored product
4. Thus, on medium containing X-gal,
white colonies contain recombinant
gene
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 Making a glowing pig

1. Isolate mRNA from jellyfish

2. Make cDNA library from this mRNA
• One of the genes in this library encodes GFP
3. Insert the cDNA library into a vector (one gene per
vector)
4. Transform the vectors into bacteria
5. Select “white colonies”, which contain inserted genes
• One vector per colony reflects that bacteria take up only
one vector during transformation
6. Amongst these white colonies, select the one that
expresses GFP
 Detection of Genes of Interest
• Colony hybridization: Sequences
complementary to the gene can be labeled
with a fluorescent tag or radioactivity and
used as a probe in hybridization
experiments to identify the clones
containing the gene.

• Colonies to be tested are transferred from a

plate onto a nitrocellulose or nylon filter.
• A part of each colony remains on the agar
medium, which constitutes the reference
plate.
• After treatment, the filter is hybridized with
labeled probe complementary in base
sequence to the gene being sought
 Making a glowing pig

1. Isolate mRNA from jellyfish

2. Make cDNA library from this mRNA
• One of the genes in this library encodes GFP
3. Insert the cDNA library into a vector (one gene per
vector)
4. Transform the vectors into bacteria
5. Select “white colonies”, which contain inserted genes
• One vector per colony reflects that bacteria take up only
one vector during transformation
6. Amongst these white colonies, select the one that
expresses GFP
7. Germ Line Transformation: introduce our cloned
GFP into the genome of pig gametes
 Germ-Line Transformation
• Germ-line transformation:
insertion of genes into the
reproductive cells of an
organism
• This produces transgenic
animals: Animals with
permanently altered genetic
• DNA is introduced into cells at
the blastocyst stage
• If gene in transformed into
cells that will differentiate into
gametes, the offspring of this
animal will contain the gene.
Another look at germ line transformation: A
knock out mouse
A knockout mouse: introducing a leptin mutation
 Applied Genetic Engineering
• Cool Pets!
• Crop plants with improved
nutritional qualities can be
created
• Animal growth rate can be
genetically engineered
• Engineered microbes can
help degrade toxic waste
• The production of useful
proteins is a primary impetus GloFish, a transgenic strain of zebrafish,
for recombinant DNA contain a red fluorescent protein from sea
anemones
Although marketed as pets, these animals
may be useful for assaying heavy metal
contamination in water
 Genetically engineered rice containing a biosynthetic
pathway for ß-carotene.
• Enzymes in the pathway derive from genes in two different
species.
• Rice plants with both parts of the pathway produce grains
with a yellowish cast because of the ß-carotene they contain

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 Alteration of Plant Genomes: Mechanism
• Recombinant DNA can also be introduced into plant genomes
• Gene transfer procedure uses Ti plasmid found in the soil
bacterium Agrobacterium tumefaciens
• Inserted genes replace a portion of plasmid and a selectable
marker
 Food production
Transgenic Atlantic salmon, grow
400–600 percent faster and have
no adverse health effects
Transgenic cows have been
developed for battling mastitis by
incorporating a gene whose protein
product destroys the S. aureus
bacterium

SciShow GMOs:
https://www.youtube.com/watch?v=sH4bi60alZU

ASAP Science:
https://www.youtube.com/watch?v=bkhhCi7nMFI
 Biomedical Applications
• Recombinant DNA technology is used to produce
large amounts of medically important proteins
• Animal viruses such as retroviruses may prove
useful vectors for gene therapy to treat single
gene disorders
• Recombinant DNA probes detect mutant genes in
hereditary disease

SciShow Human Engineering:

https://www.youtube.com/watch?v=mAmHoCbhyaw
Southern Blotting: Another look

Southern blot:
http://highered.mheducation.com/sites
/0072556781/student_view0/chapter1
4/animation_quiz_5.html
 Genomics and Proteomics
Genomics deals with the DNA sequence, organization,
function, and evolution of genomes
• made possible by the invention of techniques of recombinant
DNA, also known as gene cloning or genetic engineering
 Bioinformatics
• Human Genome Project:
sequencing of the human
genome made possible by
Rapid automated DNA
sequencing
• A genome sequence is
coded index using only imaginarymedicine.wordpress.com

four letters
• Decoding (annotating)
this index requires
identifying genes and their
functions is a major
challenge.
 Bioinformatics
• Bioinformatics, use of computers in the interpretation and
management of biological data, such as the annotation of
genomic sequences.
• Comparative genomics: using evolutionary signatures,
conserved regions, to identify significant genomic elements.

Evolutionary signatures observed among 12 Drosophila genomes

 Functional Genomics
• Functional genomics:
Identifying significant genomic
elements by examination of
genome-wide patterns of gene
expression
• DNA microarray (or chip)
• a flat surface about the size
of a postage stamp
• Up to 100,000 distinct spots
containing different
immobilized DNA sequences
• RNA or cDNA (expressed
genes) isolated from cells of
interest can be identified
based on hybridization to this
chip
Microarrays: another look

Animations:
http://highered.mcgraw-
hill.com/sites/0072556781/stud
ent_view0/chapter15/animation
_quiz_2.html
 Reverse Genetics: Loss of Function
Assays
Reverse genetics: Wildtype genes are cloned and
intentionally mutated in specific ways
• They are then introduced back into the organism
• To study the phenotypic effects of the mutations
• This a way to understand the functional importance of a
gene
 Transformation Rescue: Gain of
function assays
• Identify important regulatory sequences in a gene by
introducing them into mutant genomes.
• The experimental approach
• clone a large fragment of DNA that includes the coding
sequence for the wildtype protein
• use germ-line transformation to introduce this fragment
into the genome of an organism that contains a mutation
of a gene
• If the introduced DNA includes all regulatory sequences
necessary for correct gene expression, then the resulting
phenotype will be wildtype
• The ability of an introduced DNA to correct a mutation is
called transformation rescue, and it means that the
fragment contains all the essential regulatory sequences