Campbell and Reece
Chapter 20
 made
possible:
• Human Genome Project completed in 9 yrs (2001)
• by 2010 genomes of > 7,000 species
 DNA
formed when segments of DNA
from 2 different species are combined in
vitro (in test tube)
 useful for analyzing genes & gene
expression
 manipulation
of organisms or their
components to produce useful products
 includes selective breeding, using
bacteria in fermentation
 the
direct manipulation of genes for
practical purposes
 useful
for researcher to have just that
portion of DNA working with
• 1 gene may be as small as 1/100,000th of a
chromosome
 bacterial
plasmids: circular DNA
molecules that replicate separately from
bacterial chromosome
• used by bacterium when environment changes
 used
to cut DNA w/in short, specific
nucleotide sequences (restriction sites) 
set of dbl stranded DNA fragments with
single stranded “sticky ends”
 sticky
ends form H-bonds with sticky
ends of C’ bases from other DNA:
temporary bonds
 DNA ligase make bonds permanent:
makes covalent bonds in sugarphosphate backbone
 http://www.dnalc.org/resources/animations/
restriction.html
 http://highered.mcgraw-
hill.com/olcweb/cgi/pluginpop.cgi?it=swf::5
35::535::/sites/dl/free/0072437316/120078/bio3
7.swf::Restriction%20Endonucleases
 http://www.dnalc.org/view/15476-
Mechanism-of-Recombination-3Danimation-with-with-basic-narration.html
 name
given original plasmid
 dfn: a DNA molecule that can carry
foreign DNA into host cell & replicate
there
 bacterial plasmids mostly used because
1. readily available from suppliers
2. can insert foreign DNA in vitro  bacterial
cell
3. multiply rapidly
 http://highered.mcgraw-
hill.com/sites/0072556781/student_view0/
chapter14/animation_quiz_2.html
 large
plasmids trimmed down so they
contain just the genes necessary for
replication
 carry 100 – 300 kb (kilo base pairs),
normal plasmid can insert vectors no
larger than 10 kb
can make “libraries” using cDNA:
 each
have advantages
 Genomic library good if:
• looking for a gene but not sure where it is in a
genome, or what kind of cell to look in
• if looking for introns or regulatory sequences
as’c w/gene
 cDNA
library good if studying:
• specific protein
• sets of genes expressed in particular cell types
• changes in patterns of genes over life of cell
(during development of organism)
 Nucleic
Acid hybridization: process of
base pairing between a gene & a C’
sequence on another nucleic acid
molecule
 C’ molecule = ssDNA or ssRNA = nucleic
acid probe
 probe is made that is C’ to known
sequence in gene
 several
technical difficulties hinder the
expression of cloned eukaryotic genes in
bacterial hosts
 can substitute eukaryotic hosts: yeasts,
some insect cell, some mammalian cells
that have appropriate expression vectors:
a cloning vector that contains a highly active bacterial
promoter just upstream of restriction site where
eukaryotic gene can be inserted allowing gene to be
expressed in bacterial cell or have been genetically
engineered to use in specific eukaryotic cells
 Polymerase
Chain Reaction
 amplifies specific target segment of DNA
in vitro using primers that bracket the
derived sequence, & a heat-resistant
DNA polymerase, & nucleotides
 http://www.dnalc.org/view/15475-The-
cycles-of-the-polymerase-chain-reactionPCR-3D-animation-with-no-audio.html
 once
you have many copies of a gene you
can ask questions about its functions,
where & when the gene is expressed, or
how important is it to the organism
 uses
a gel made of a polymer (agarose
commonly)
 gel acts like molecular sieve
 separates nucleic acids or proteins based
on charge and size
 nucleic acids carry (-) charges on
phosphate group so  (+) end of gel
 as move the longer molecules impeded
more by “sieve”
 fragments
produced be restriction
enzymes put thru gel electrophoresis 
band pattern characteristic of starting
molecule & restriction enzyme used
 able to identify viruses & plasmids by
their band patterns
 then recover DNA from gels
 1 way of getting pure samples of DNA –
won’t work with DNA from eukaryotic
cells: gel electrophoresis  smear not
bands
 Restriction
Fragment Length
Polymorphism: single nucleotide
polymorphism (SNP) that exists in the
restriction site for a particular enzyme,
making the site unrecognizable by that
enzyme & changing lengths of the
restriction fragments formed by digestion
with that enzyme
 found in coding & noncoding DNA
 used
to detect certain nucleotide
sequences w/in a complex DNA sample
 compares the restriction fragments
produced from different samples of
genome DNA
 method
used to sequence relatively short
DNA fragments
 done by automated sequencing machines
 technique:
synthesizes a set of DNA
strands C’ to original DNA fragment
• each strand starts with same primer & ends with
dideoxyribonucleotide (ddNTP)
• incorporation of ddNTP terminates growing
DNA strand because it lacks 3’ –OH group (site
of attachment of next nucleotide)
• each ddNTP tagged with distinct fluorescent
label so identity of nucleotide at end of each
strand (ultimately entire strand) is identified
 in
vitro hybridization with labeled
probes looking for specific mRNAs
 could be used to look at how expression
of a gene changes during the embryonic
development of organism
 carry out gel electrophoresis on mRNA
 Reverse
Transcriptase- Polymerase Chain
Reaction
• quicker & more sensitive than Northern blotting
• isolates mRNA from different developmental
stages of organism then add reverse transcriptase
to make cDNA which serves as template for PCR
amplification using primers from gene being
studied
• bands will be in samples that originally
contained the gene being studied
 alternative
method used to determine
which cells are expressing certain genes
 done in living organism
 probes labeled with fluorescent dyes
 uncover
gene interactions
 suggest correct therapeutic route in
cancer treatments
 most
common method used to determine
function of gene: disable it & observe
what happens
 specific mutations introduced to cloned
gene & then mutated gene returned to
cell knocking out normal gene in the
process
 RNA
interference
 method for silencing expression of
selected genes
 uses synthetic dsRNA molecules
matching the sequence of gene to trigger
breakdown of the gene’s mRNA or to
block its translation
 important
when studying groups of
genes to determine how multiple genes
interact (basis of systems biology, chap
21)
 in humans considered unethical to block
activity of genes
 used
to analyze genomes of large #s of
humans with certain phenotype or
disease
 test for genetic markers: DNA sequences
that vary in a population
• uses SNPs (single nucleotide polymorphisms)
• single base pair site where variation is found in
at least 1% of population
• few million in human genome
 as
advances being made in DNA
technology also working on technology
to make multicellular organism from 1
cell producing genetically identical
organism
 1st attempted late 1950’s
 Genomic
Equivalence: an organism’s
cells have the same genome
• proved when able to generate new organism
from 1 cell
• used carrot (root) cells  cultured  adult plant
 totipotent:
describing a cell that can give
rise to all parts of the embryo & adult, as
well as extraembryonic membranes in
species that have them
 used
in cloning animals
 transplant nucleus from a differentiated
animal cell  enucleated ova can
sometimes give rise to clone
 Embryonic
Stem Cells (ES) or Adult Stem
Cells from animal embryos or adult
tissues can reproduce & differentiate in
vitro and in situ
 ES cells are pluripotent: cell that can give
rise to many but not all parts of an
organism
• difficult to acquire
 ES
cells currently donated by patients
undergoing infertility treatment or from
long term cell cultures established with
cells isolated from donated embryos
 when
main objective is to produce ES to
treat disease process called therapeutic
cloning
 now
scientists can “de-differentiate” cells
returning them to pluripotent cells: called
iPS: induces Pluripotent stem cells
• can do anything ES cells can do
2
1.
major uses
reprogram cells from patients with
disease to become iPS cells
•
•
2.
then act as model cells for studying the disease
& potential treatments
Parkinson’s disease, type 1 diabetes
field of regenerative medicine
•
patient‘s own cells used to regenerate damaged
tissues
 introducing
genes into afflicted
individual (into somatic cells) for
therapeutic purposes
 useful for disorders caused by single
gene defect (overall, relatively small # of
all diseases)
 for it to be permanent, treated cells must
be the ones that continue to divide thru
out patient’s life
 using
Short Tandem Repeats (STRs) in
DNA isolated from crime scenes leads to
genetic profile
• strong evidence to prove suspect innocent or
guilty
• used in paternity disputes
• identification of remains
 gene
for desired protein inserted into
bacterial genome and become tiny
“factory” for making protein
• Insulin
• Digestive enzymes
• Growth Hormone
 genetically
engineered microorganisms
developed for oil spills or to degrade
toxic waste materials
• Bacteria
• Algae
• Plants
 to
improve productivity & food quality