Download Chapter 20 Notes: DNA Technology

Chapter 20 Notes:
DNA Technology
Understanding & Manipulating Genomes
• 1995: sequencing of the first complete genome
(bacteria)
• 2003: sequencing of the Human Genome mostly
completed
• These accomplishments depended on new technology:
– Recombinant DNA: DNA from 2 sources (often 2 species)
are combined in vitro into the same DNA molecule
• Called Genetic engineering: direct manipulation of genes for
practical purposes
 DNA technology has launched
a revolution in the are of:
 BIOTECHNOLOGY: the
use of living organisms or their
components to do practical
tasks
-microorganisms to make
wine/cheese
-selective breeding of
livestock
-production of antibiotics
-agriculture
-criminal law
**Practical goal of biotech =
improvement of human health
and food production
Ch 20 looks at:
1. Main techniques for manipulating DNA
2. How genomes are analyzed & compared
at the DNA level
3. Practical applications of DNA technology
(including social & ethical issues)
“Toolkit” for DNA technology involves:
-DNA vectors
-host organisms
- restriction enzymes
VECTORS = carriers for moving DNA
from test tubes back into cells
-bacterial plasmids (small, circular
DNA molecules that replicate within
bacterial cells)
-viruses
HOST ORGANISMS:
bacteria are commonly
used as hosts in genetic
engineering because:
1) DNA can easily be isolated from
& reintroduced into bacterial cells;
2) bacterial cultures grow quickly,
rapidly replicating any foreign
genes they carry.
RESTRICTION ENZYMES = enzymes that
recognize and cut short, specific nucleotide
sequences (called restriction sites)
-in nature, these enzymes protect the
bacterial cell from other organisms by
cutting up their foreign DNA
Restriction Enzymes (cont.)…
most restriction sequences are symmetrical
in that the same sequence of 4-8
nucleotides is found on both strands, but
run in opposite directions
restriction enzymes usually cut
phosphodiester bonds of both strands in a
staggered manner producing single
stranded “sticky ends”
Restriction Enzymes (cont.)…
“sticky ends” of restriction
fragments are used in the
lab to join DNA pieces
from different sources
(complementary base
pairing)
*RECOMBINANT DNA
unions of different DNA
sources can be made
permanent by adding DNA
ligase enzyme (form
covalent bonds between
bases)
DNA Technologies:
1) Cloning
2) DNA fingerprinting
(profiling)
3) Microarray
4) Gene therapy
Steps Involved in
Cloning a Human Gene:
Human gene
plasmid
1) Isolate human gene to clone (ex: insulin);
2) Isolate plasmid from bacterial cell;
3) cut both DNA samples with the same
restriction enzyme to open up bacterial
plasmid & create sticky ends on bath
samples;
4) Mix the cut plasmids and human DNA genes
& seal with DNA ligase;
Cloning a Human Gene (cont.)…
5) Insert recombinant DNA plasmid back into
bacterial cell;
6) As bacterial cell reproduces, it makes copies
of the desired gene;
-grow cells on a petri dish
7) Identify cell clones carrying the gene of
interest.
-HOW? Which ones took up the gene & are
making insulin?
-Add a 2nd gene besides insulin; add one for antibiotic
resistance & then grow bacteria on that antibiotic
LE 20-4_3
Bacterial cell
Isolate plasmid DNA
and human DNA.
lacZ gene
(lactose
breakdown)
Human
cell
Restriction
site
ampR gene
(ampicillin
resistance)
Cut both DNA samples with
the same restriction enzyme.
Bacterial
plasmid
Gene of
interest
Sticky
ends
Human DNA
fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Recombinant DNA plasmids
Introduce the DNA into bacterial cells
that have a mutation in their own lacZ
gene.
Recombinant
bacteria
Plate the bacteria on agar
containing ampicillin and X-gal.
Incubate until colonies grow.
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying
recombinant
plasmid with
disrupted lacZ gene
Bacterial
clone
Why can bacteria produce insulin through recombinant
DNA technology? The genetic code is universal!!!!
DNA Fingerprinting Involves: The
Polymerase Chain Reaction (PCR)
 allows any piece of DNA to be quickly
amplified (copied many times) in vitro
(artificial environment).
 DNA is incubated under
appropriate conditions
with special primers &
DNA polymerase
molecules
PCR (continued)…
 BILLIONS of copies of
DNA are produced in just a
few hours (enough to use
for testing)
In 6 cycles of PCR:
cycle 1: 2 copies
cycle 2: 4 copies
cycle 3: 8 copies
cycle 4: 16 copies
cycle 5: 32 copies
cycle 6: 64 copies
cycle 20: 1,048,576!!
Polymerase Chain Reaction (PCR)
PCR is highly specific;
primers determine
the DNA sequence to
be amplified
 only minute amounts
of DNA are needed
Remember
these?
Starting materials
for PCR:
• DNA to be copied
• Nucleotides
• Primers
• Taq polymerase
(DNA polymerase isolated from
bacteria living in hot springs…their
enzymes can withstand high temps!)
Steps of PCR:
1) Heat to separate
DNA strands (95ºC);
2) Cool to allow primers
to bind (55ºC);
3) Heat slightly so that
DNA polymerase
extends the 3’ end of
each primer (72ºC)
4) Repeat steps #1-3
many times!!!
Applications of PCR:
 DNA from tiny amounts of tissue or semen
found at crime scene;
 DNA from single embryonic cells for
prenatal diagnosis;
 DNA or viral genes from cells infected
with difficult to detect viruses such as
HIV;
 used extensively in Human Genome Project
to produce linkage maps without the need
for large family pedigree analysis.
PCR works
like a
copying
machine for
DNA!
Analysis of Cloned DNA:
Gel electrophoresis
 separates macromolecules (nucleic acids or
proteins) based on size, charge, or other
property (DNA cut with restriction enzymes)
 for linear molecules like DNA, separation
depends mainly on size
 a mixture of DNA fragments will be sorted
into bands, each consisting of DNA molecules
of the same length
YOUR DNA
MY DNA
-
+
The DNA (-) is
loaded at the top in
a well
Steps Involved in DNA Fingerprinting:
1) Collect DNA from a sample;
2) Perform PCR if necessary to
make more DNA;
3) Cut DNA apart using RE’s
which will recognize specific base
sequences
**DNA will be cut at different places for
different people, therefore producing different
size fragments
(Restriction Fragment Length Polymorphisms…
a.k.a. RFLP’s- noncoding regions between genes)
DNA Fingerprinting
(cont.)…
4) Electrophoresis is used to align DNA pieces
on gel called agarose; electric current is
used to actually move DNA pieces across
the gel;
5)DNA fragments are chemically separated and
transferred from the gel to a sheet of nylon and
which is then exposed to radioactive probes which
will stick to DNA complementary base pairs;
1 method of visualizing
the DNA
DNA Fingerprinting (cont.)…
6) Nylon sheet is placed against x-ray film
and developed; when 2 bases join, a black
band will appear;
7) Banding patterns can then be compared.
Triangles: RE cut site
“a” allele lost cleavage site
#2 due to a mutation; results
in a longer fragment
Dark line represents a probe.
Analysis and
inheritance of
allelic RFLP
fragments (NIH).
Sample Sample
1
2
DNA_DetectivePC.exe
DNA Microarray Assays
• Technique used to study how genes act together to
produce & maintain a functioning organism
– Which genes are transcribed in different situations such as
different tissues or at different stages of development
• Consists of tiny amounts of a large number of single
strand-stranded DNA fragments representing different
genes fixed to a glass slide in a tightly spaced array
(grid)
– Aka: DNA chip
Microarray Technique
1. Isolate mRNA
2. Make cDNA by reverse transcription, using
fluorescently labeled nucleotides
3. Apply the cDNA mixture to a microarray; the
cDNA will hybridize with any complementary
DNA on the microarray
4. Rinse off excess cDNA & scan for fluorescence
•
•
each fluorescent spot represents a gene being
expressed
The intensity of fluorescence indicates level of
expression
LE 20-14
Tissue sample
Isolate mRNA.
Make cDNA by reverse
transcription, using
fluorescently labeled
nucleotides.
Apply the cDNA mixture to a
microarray, a microscope slide
on which copies of singlestranded DNA fragments from
the organism’s genes are fixed,
a different gene in each spot.
The cDNA hybridizes with any
complementary DNA on the
microarray.
Rinse off excess cDNA; scan
microarray for fluorescent.
Each fluorescent spot
(yellow) represents a gene
expressed in the tissue
sample.
mRNA molecules
Labeled cDNA molecules
(single strands)
DNA
microarray
Size of an actual
DNA microarray
with all the genes
of yeast (6,400 spots)
Practical
Applications of DNA
Microarrays
• Track gene expression
changes from initial
development throughout an
organisms lifetime
• Contribute to better
understanding of certain
diseases & suggest new
diagnostic techniques or
therapies
Gene Therapy
•Alteration of an afflicted
individual’s genes
•Experimental: in theory,a
normal allele of a defective gene
could be inserted into the
somatic cells of the tissue
affected by the disorder
•Somatic cells must multiply
throughout patients life (ex:
bone marrow cells)
LE 20-16
Cloned gene
Insert RNA version of normal allele
into retrovirus.
Viral RNA
Retrovirus
capsid
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
Inject engineered
cells into patient.
Bone
marrow
Practical Applications of DNA
Technology:
Medicine / Pharmaceutical
1) human gene therapy
2) pharmaceutical products
-insulin, growth hormone, TPA (dissolves
blood clots), proteins that mimic cell surface
receptors for viruses like HIV
3) diagnosis of disease
LE 20-9
Normal -globin allele
175 bp
Ddel
201 bp
Ddel
Large fragment
Ddel
Ddel
Sickle-cell mutant -globin allele
376 bp
Ddel
Large fragment
Ddel
Ddel
Ddel restriction sites in normal and sickle-cell alleles of
-globin gene
Normal
allele
Sickle-cell
allele
Large
fragment
376 bp
201 bp
175 bp
Electrophoresis of restriction fragments from normal
and sickle-cell alleles
Applications of DNA
Technology…
Forensic uses
DNA fingerprints: paternity, criminal
cases
 Environmental uses: microorganisms
engineered to break down sewage, oil
spills, etc.
Applications of DNA
Technology…
 Agricultural uses (“Pharm” Animals and Plants)
1) livestock
-bGH (bovine growth hormone) to enhance milk production
-sheep that carry a gene for human blood protein
-genes that cause devp’t of larger muscles in cattle
-salmon that grow larger due to a modified growth hormone
2) genetically engineered plants
- insecticide/herbicide resistance
- decaying of ripening/resistance to spoiling
- transgenic rice that contain beta-carotene to solve the
vitamin A deficiency in poorer countries
Regular Tomato
Flavor Savr Tomato
LE 20-19
Agrobacterium tumefaciens
Ti
plasmid
Site where
restriction
enzyme cuts
T DNA
DNA with
the gene
of interest
Recombinant
Ti plasmid
Plant with
new trait
Why is this a restriction enzyme site?
A. Restriction enzymes bind to
special hydrogen bond sites
B. Restriction enzymes cut at GAATTC
C. Restriction enzymes cut at CTTAAG
D. Restriction enzymes recognize specific reverse order
sequences
What are “sticky ends?”
A.
B.
C.
D.
Pieces of DNA that are run through a gel in order to give a
unique banding pattern
Single-stranded DNA ends that are available to hydrogen bond
to a complimentary single strand
DNA bases that are added to a PCR machine so that multiple
exact copies of a DNA sequence can be produced
Segments of DNA that act as a probe in order to diagnose a
genetic condition
What is this called?
A.
B.
C.
D.
RFLP (restriction length polymorphism)
PCR (polymerase chain reaction)
Clone
Recombinant DNA
plasmid
Human gene
What is the purpose of PCR?
A.
B.
C.
D.
To make billions of identical copies of a specific DNA segment
To separate out DNA segments by size
To help make medical products such as insulin
To help cure diseases such as Cystic Fibrosis
What is the basis for DNA movement in gel electrophoresis?
A. DNA has a (+) charge so it moves towards a (-) electrical
terminal
B. DNA has a (+) charge so it moves towards a (+) electrical
terminal
C. DNA has a (-) charge so it moves towards a (+) electrical
terminal
D. DNA has a (-) charge so it moves towards a (-) electrical
terminal
Which of the following is NOT an application of
biotechnology?
A. Agricultural products such as bovine growth hormone
B. Pharmaceutical products such as insulin
C. Environmental uses; cleaning up oil spills
D. Medical uses; to help parents have children with specific
traits
E. Medical uses; to help diagnose some diseases
Vocabulary terms
Transcription is the process of synthesizing
RNA using a DNA duplex as template.
Translation is the process of synthesizing a
protein using an mRNA molecule as a guide.
Gene expression refers to the transcription
and translation of a gene or set of genes.
Gene regulation refers to the control of
gene expression.
more vocab…
Hybridization is the process by which two
complementary strands of nucleic acid base
pair to one another to form a duplex. If
two strands of nucleic acid are not
complementary, they will not hybridize to
form a duplex.
Gene knockouts are experiments in which a
gene is deleted from the genome of an
organism. Knockouts are used to gain
information about the function of a gene.
still more vocab…
Transcriptional regulation is the
control of gene expression at the
level of transcription.
RNA interference (RNAi) is the
phenomenon in which experimentally
introduced double-stranded RNA
leads to loss of expression of the
corresponding cellular gene.