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Electrophoresis made Easy
Biotechnology
Chapter 13 2
Traditional Applications
Biotechnology is applied biology
• Modern focus on genetic engineering,
recombinant DNA technology, and analysis of
biomolecules
Chapter 13 3
Traditional Applications
Traditional (historical) applications of
biotechnology date back to over 10,000
years ago
• Use of yeast to produce beer and wine in Egypt
and Near East
• Selective breeding of plants
• Selective breeding of animals
Chapter 13 4
Genetic Engineering
Genetic engineering refers to the modification
of genetic material to achieve specific
goals
• Learn more about cellular processes,
including inheritance and gene expression
• Provide better understanding and treatment
of diseases, particularly genetic disorders
• Generate economic and social benefits
through production of valuable biomolecules
and improved plants and animals for
agriculture
Chapter 13 5
Recombinant DNA
Genetic engineering utilizes recombinant DNA
technology
• Splicing together of genes or portions of
genes from different organisms
Recombinant DNA can be transferred to
plants and animals
• Modified animals are called transgenic or
genetically modified organisms (GMOs)
• Most modern biotechnology includes
manipulation of DNA
Chapter 13 6
Recombination in Nature
Many natural processes recombine DNA:
Due to crossing over during meiosis, each
chromosome in a gamete contains a
mixture of alleles from the two parental
chromosomes
• Thus, eggs and sperm contain recombinant
DNA
Chapter 13 7
Transformation
Bacteria can naturally take up DNA from the
environment (transformation) and integrate
the new genes into the genome
(recombination)
Recombination Chapter 13 8
Plasmid DNA fragments
in Bacteria transferred transferred
(a) Bacterium (b) to new host(c) to new host
Chromosome
Plasmid
1 µm Plasmid
replicates in
cytoplasm DNA fragment
incorporated
into
chromosome
Chapter 13 9
Transformation
Small circular DNA molecules (plasmids) carry
supplementary genes
• Plasmid genes may allow bacteria to grow in
novel environments
• Plasmid genes may enhance virulence of
bacteria in establishing an infection
• Plasmid genes may confer resistance to
antimicrobial drugs
Chapter 13 10
Viral Transfer of DNA
Viral life cycle
1. Viral particle invades host cell
2. Viral DNA is replicated
3. Viral protein molecules are synthesized
4. Offspring viruses are assembled and break
out of the host cell
Chapter 13 11
Viral Transfer of DNA
Viral transfer of DNA
• Viruses may package some genes from host
cell into viral particles during assembly
• Infection of new host cell injects genes from
previous host, allowing for recombination
Chapter 13 12
Viruses May Transfer Genes
Chapter 13 13
Biotechnology and Forensics
Forensics is the science of criminal and victim
identification
DNA technology has allowed forensic science
to identify victims and criminals from trace
biological samples
• Genetic sequences of any human individual are
unique
• DNA analysis reveals patterns that identify
people with a high degree of accuracy
Chapter 13 14
Polymerase Chain Reaction
Forensic technicians typically have very little
DNA with which to perform analyses
Polymerase Chain Reaction (PCR) produces
virtually unlimited copies of a very small
DNA sample
Chapter 13 15
Polymerase Chain Reaction
PCR requires small pieces of DNA (called
primers) that are complementary to the
gene sequences targeted for copying
A PCR “run” is basically DNA replication in a
tiny test tube
• Template DNA, primer, nucleotides, and DNA
polymerase are all in the reaction mix
Chapter 13 16
Polymerase Chain Reaction
Four steps of a PCR cycle
1. Template strand separation
– The test tube is heated to 90-95oC to cause the
double stranded template DNA to separate into
single strands…
2. Binding of the primers
– The temperature is lowered to 50oC to allow the
primer DNA segments to bind to the targeted gene
sequences through hydrogen bonding…
Chapter 13 17
Polymerase Chain Reaction
3. New DNA synthesis at targeted sequences
The temperature is raised to 70-72oC where the
heat-stable DNA polymerase synthesizes new
DNA of the sequences targeted by the
primers…
4. Repetition of the cycle
The cycle is repeated automatically (by a
thermocycler machine) for 20-30 cycles,
producing up to 1 billion copies of the original
targeted DNA sequence
Polymerase Chain Reaction: Chapter 13 18
(a) One PCR Cycle
90 °C 50 °C 72 °C
DNA Polymerase Primer
DNA
Original Separate Primers & DNA
Double- DNA DNA synthesized
helix Strands polymerase
DNA bind
Polymerase Chain Reaction: Chapter 13 19
(b) Multiple PCR Cycles
2 copies 4 copies 8 copies
DNA
fragment
to be
amplified
Chapter 13 20
Polymerase Chain Reaction
Choice of primers determines which
sequences are amplified (copied)
Forensic scientists focus on short tandem
repeats (STRs) found within the human
genome
Chapter 13 21
Polymerase Chain Reaction
STRs are repeated sequences of DNA within
the chromosomes that do not code for
proteins
STRs vary greatly between different human
individuals
A match of 10 different STRs between
suspect and crime scene DNA virtually
proves the suspect was at the crime scene
Chapter 13 22
Chapter 13 23
Gel Electrophoresis
Mixtures of DNA fragments can be separated
on the basis of size
Gel electrophoresis is a technique used to
spread out different-length DNA fragments
in a mixture
Chapter 13 24
Gel Electrophoresis
Four steps of gel electrophoresis
1. DNA mixtures are placed into wells at one
end of a slab of agarose gel
2. An electric current introduced through the
gel causes the negatively-charged DNA
fragments to migrate towards the positive
electrode
Chapter 13 25
Gel Electrophoresis
Four steps of gel electrophoresis
3. Short DNA fragments move more easily
through the three-dimensional meshwork of
fibers between the gel
Short DNA fragments migrate farther than
long DNA fragments so the mixture is
separated into bands of DNA of specific
lengths
4. The invisible bands of DNA are made visible
using stains or DNA probes
Chapter 13 26
Chapter 13 27
Chapter 13 28
RFLP: Gel Electrophoresis
Larger fragments
Direction move more slowly;
of Migration smaller fragments
move more rapidly
Chapter 13 29
DNA Fingerprinting
DNA from a crime scene sample can be
amplified by PCR and run on a gel with
suspect DNAs
Short tandem repeats (STRs) in the gel DNA
can be identified by DNA probes
Distinctive pattern of STR numbers and
lengths are fairly unique to a specific
individual (forming a DNA fingerprint)
DNA fingerprint from crime scene can be
matched with DNA fingerprint of suspect
Chapter 13 30
DNA Fingerprint in Forensics
Q: Which suspect A: #3 is prime
should be suspect
indicted?
1 2 3 CS 4 5 67
RC
IE
Suspects MN Suspects
EE
Chapter 13 31
Restriction Enzymes Cut DNA
A DNA sequence (e.g. a gene) can be removed
from a chromosome using special enzymes
Restriction enzymes are nucleases that cut DNA
at specific nucleotide sequences
Chapter 13 32
Chapter 13 33
Restriction Enzymes Cut DNA
Enzymes that create staggered cuts with “sticky
ends” are the most useful in gene cloning
Chapter 13 34
Splicing of DNA Fragments
Sticky ends allow for splicing of a DNA
fragment with another complementary
fragment (Bt – Bacillus thurengensis)
• Bt gene can be cut out of the Bacillus
chromosome with the same enzyme used to
cut open the plasmid
• Bt gene fragment ends can base-pair with
sticky ends of the opened plasmid, adding
gene to the plasmid circle
Chapter 13 35
Splicing of DNA Fragments
DNA ligase enzyme used next to permanently
bond gene into plasmid
Chapter 13 36
Chapter 13 37
Chapter 13 38
Chapter 13 39
Chapter 13 40
Chapter 13 41
Chapter 13 42
Plasmids Are Used to Insert Genes
The Ti plasmid from Agrobacterium
tumefaciens is ideal for transferring genes
into plant chromosomes
Chapter 13 43
Plasmids Are Used to Insert Genes
Agrobacterium infects plant cells and inserts
its small Ti plasmid into a plant
chromosome in the nucleus
• Pathogenic effects of certain tumor-causing Ti
plasmid genes can be disabled
• A gene inserted into a Ti plasmid is therefore
carried into the plant cell chromosomes by a
natural process
Chapter 13 44
Chapter 13 45
The Human Genome Project
Findings
• Human genome contains ~25,000 genes
• New genes, including many disease-associated
genes have been discovered
• Has determined the nucleotide sequence of all
the DNA in our entire set of genes, called the
human genome
• The genes comprise 2% of all the DNA
Chapter 13 46
The Human Genome Project
Applications
• Improved diagnosis, treatment and cures of
genetic disorders or predispositions
• Comparison of our genome to those of other
species will clarify the genetic differences that
help to make us human
Chapter 13 47
DNA Probes
Defective alleles can also be identified using
DNA probes
DNA probing is especially useful where there
are many different alleles at a single gene
locus
• Cystic fibrosis is a disease caused by any of
32 alleles out of 1000 total possible alleles
Chapter 13 48
DNA Probes
Arrays of single-stranded DNA complementary
to each of the defective alleles can be bound
to filter paper
A person’s DNA sample is cut up and separated into
single-strands
The array is bathed in the DNA sample
Strands of DNA binding to complementary sequence
on the paper indicate presence of a defective
allele in person’s genome
Chapter 13 49
Chapter 13 50
DNA Probes
An expanded version of this type of DNA analysis is
known as a microarray
A microarray contains up to thousands of probes for
a variety of disease-related alleles
Microarray analysis has the potential to
comprehensively identify disease susceptibility
Chapter 13 51
Chapter 13 52
Scientific Objections to GMOs
Safety issues from eating GMOs
• Could ingestion of Bt protein in insect-
resistant plants be dangerous to humans?
• Are transgenic fish producing extra growth
hormone dangerous to eat?
• Could GM crops cause allergic reactions?
– USDA now monitors GM foods for allergic
potential
• Toxicology study of GM plants (2003)
concluded that ingestion of current transgenic
crops pose no significant health dangers
Chapter 13 53
Scientific Objections to GMOs
Environmental hazards posed by GMOs
• Pollen from modified plants can carry GM
genes to the wild plant population
– Could herbicide resistance genes be transferred
to weed species, creating superweeds?
• Could GM fish reduce biodiversity in the wild
population if they escape?
– Reduced diversity in wild fish makes them more
susceptible to catastrophic disease outbreaks
Chapter 13 54
Scientific Objections to GMOs
Environmental hazards posed by GMOs
• US found to lack adequate system to monitor
changes in ecosystem wrought by GMOs
(National Academy of Science Study 2003)
Chapter 13 55
The Human Genome
Should parents be given information about the
genetic health of an unborn fetus?
Should parents be allowed to select the
genomes of their offspring?
• Embryos from in vitro fertilization are
currently tested before implantation
• Many unused embryos are discarded
Should parents be allowed to design or
correct the genomes of their offspring?
Chapter 13 56
Hope through Gene Therapy
Human Cloning: Chapter 13 57
Permanent Genetic Correction?
Parents with Zygote with Baby with
genetic disease defective gene genetic disorder
Viral
vector
with
therapeutic
gene Cell Culture Embryo with
defective gene
Treated Culture Genetically
corrected
Enucleated Genetically embryo clone
egg cell corrected
egg cell
Genetically corrected
cell from culture Healthy baby
Chapter 13
The end
see also the following related videos:
1. Plasmid Cloning
2. DNA Fingerprinting
3. Gene Therapy
and many more!
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