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BIOLOGY TENTH
EDITION
Lecture 9
(based on Chapter19)
DNA Technology
The DNA toolbox
a) X ray crystallography
b) Nuclear magnetic resonance (NMR) spectroscopy, which does not
require protein crystallization
c) Bioinformatics uses computer programs to predict protein structure
from amino acid sequences
X ray crystallography
EXPERIMENT
Diffracted
X-rays
X-ray
source X-ray
beam
RNA
polymerase II
DNA
RNA
Concept 19.1: DNA sequencing and DNA cloning are valuable tools for
genetic engineering and biological inquiry
Recombinant
bacterium
3 Host cell grown in culture to form a clone of
cells containing the “cloned” gene of interest
Gene of
interest Protein expressed
from gene of interest
4 Basic research
Gene for pest resistance Human growth hormone
and various
inserted into plants treats stunted growth
applications
a) The most useful restriction enzymes cut DNA in a staggered way to form
‘sticky ends’ that will attach to sticky ends of other fragments.
Restriction site
5′ 3′
G AAT T C
DNA
C T T AAG
3′ 5′
1 Restriction enzyme cuts
the sugar-phosphate dna ligase
backbones at each arrow.
3′ 5′
5′ 3′
5′ 3′
3′ 5′
Sticky end
5′
3′
3′
5′
Fragment from different DNA molecule
cut by the same restriction enzyme
5′ 3′ 5′ 3′ 5′ 3′
G AAT T C G AAT T C
C T TA A G C T TAA G
3′ 5′ 3′ 5′ 3′ 5′
One possible combination
3 DNA ligase
seals the strands.
insulin
5′ 3′
Recombinant
plasmid
Animation: Restriction Enzymes
a) To separate and visualize the fragments produced, gel electrophoresis
would be carried out
b) This technique uses a gel made of a polymer to separate a mixture of
nucleic acids or proteins based on size, charge, or other physical
properties
Figure 19.7
Mixture of Power
DNA mol- source
ecules of Cathode Anode
different
sizes
Wells
Gel
Restriction fragments
(size standards)
(b) Shorter molecules are slowed down less than
longer ones, so they move faster through the gel.
Amplifying DNA: The Polymerase Chain Reaction (PCR) and Its Use in
DNA Cloning
3′ 5′
PCR produces a 2 Annealing
Cycle 1
chain reaction yields
Primers
2
that produces an molecules
3 Extension
exponentially New
growing nucleotides
population of
identical DNA Cycle 2
yields
molecules 4
molecules
Cycle 3 yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
Results After 30 more cycles, over 1 billion (109) molecules match the target sequence.
Animation: Cloning a Gene
Reverse transcriptase-polymerase chain reaction DNA in
(RT-PCR) nucleus
mRNAs in
cytoplasm
5′ 3′
3′ 5′
cDNA
Technique
1 cDNA synthesis mRNAs
cDNAs
Primers
2 PCR amplification
Specific
gene
3 Gel electrophoresis
wg mRNA en mRNA
Cells Cells
expressing expressing
the wg gene the en gene
Head Thorax Abdomen
50 µm T1 T2 T3 A1 A2 A3 A4 A5
Studying the Expression of Interacting Groups of Genes
Genes expressed
in second tissue.
Genes expressed
in both tissues.
Genes expressed
► in neither tissue.
DNA microarray
(actual size)
DNA microarray
a) One way to determine function is to disable the gene and observe the
consequences
b) Using in vitro mutagenesis, mutations are introduced into a cloned gene,
altering or destroying its function
c) When the mutated gene is returned to the cell,
the normal gene’s function might be determined by examining the mutant’s
phenotype
Determining Gene Function
a) These are single nucleotide variants and are useful genetic markers
b) Scientists analyze the genomes of many people with a certain genetic
condition to try to find nucleotide changes specific to the condition
c) SNP variants that are found frequently associated with a particular inherited
disorder alert researchers to the most likely location for the disease-causing
gene
d) SNPs are rarely directly involved in the disease; they are most often in
noncoding regions of the genome
A
DNA
T
Normal allele
SNP
C
G
Disease-causing
allele
Concept 19.3: Cloned organisms and stem cells are useful for basic
research and other applications
agriculture
Cloning Plants: Single-Cell Cultures
a) In plants, cells can dedifferentiate and then give rise to all the specialized
cell types of the organism
b) A totipotent cell is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
The cloning of a whole carrot plant from a single carrot cell
Cross section
of carrot root
Small
fragments
Fully differ-
Less differ- entiated
entiated cell (intestinal) cell
Donor Donor
nucleus Enucleated nucleus
trans- egg cell trans-
planted Egg with donor nucleus planted
activated to begin
Results development
1 2
The groundbreaking Egg cell
technique used from ovary Nucleus
removed
involved transferring Cultured
mammary
3 Cells fused
Surrogate
mother
6 Embryonic
development
Lamb (“Dolly”)
Results genetically identical to
mammary cell donor
Technique
1 2
An electric shock Egg cell
stimulated the hybrid from ovary Nucleus
removed
cell to begin dividing Cultured
mammary
3 Cells fused
Surrogate
mother
6 Embryonic
development
Lamb (“Dolly”)
Results genetically identical to
mammary cell donor
Reproductive Cloning of Mammals
a) Stem cells are relatively unspecialized cells that can both reproduce
indefinitely and, under certain conditions, differentiate into one or more
specialized cell types
Stem cell derived from the bone marrow
Cell division
Different culture
conditions
Different types of
differentiated cells
ethical issues
a) Researchers can treat differentiated cells, and reprogram them to act like
ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell
master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions of
ES cells
d) iPS cells can be used as models for study of certain diseases and
potentially as replacement cells for patients
Concept 19.4: The practical applications of DNA-based biotechnology
affect our lives in many ways
Viral RNA
basic
3 Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient