Transcript Chapter 20 Notes: DNA Technology

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.