Download ENVIRONMENTAL CHALLENGES

GENETIC TECHNOLOGY
Chapter 14
A. Manipulation and Modification of
DNA
1. Restriction Enzymes
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Recognize specific sequences of DNA
(usually palindromes) and make cuts.
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These cuts usually produce “sticky ends”
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Used to remove segments of
DNA which can be “pasted” into
another piece of DNA using the
enzyme ligase.
2. Reverse Transcriptase
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An enzyme produced by retroviruses
Creates a double stranded DNA molecule
from an RNA template
Complementary DNA (cDNA) is produced
when reverse transcriptase creates
DNA from purified mRNA
• cDNA lacks introns
3. Vectors
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Molecules that maintain a DNA molecule
of interest and carry it into a cell
Plasmids (extra pieces of DNA found in
bacteria) and retroviruses are common
vectors.
B. DNA Amplification
1. Polymerase chain reaction (PCR)
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Used to produce millions of copies of a
target DNA sequence within hours
Requires the following items which are
subjected to cycles of heating and
cooling:
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A target DNA sequence
Primers
DNA nucleotides
Heat stable DNA polymerase (Taq)
C. Separation and Analysis of DNA
1. Gel electrophoresis
DNA fragments of various sizes are
loaded into an agarose gel where an
electrical current is applied.
• DNA has a negative charge and
migrates towards a positive charge.
Smaller DNA fragments are able to
move faster than larger fragments.
• After staining, a banding pattern is
visible.
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2. DNA Fingerprints
Patterns produced when DNA is cut by
restriction enzymes
Produced by looking at:
• SNPs (single nucleotide
polymorphisms) - highly variable
regions of genomes which vary from
one individual to the next
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SNPs can be cut with restriction
enzymes which will produce different
sized DNA fragments in different
individuals.
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Comparison of repeated DNA
sequences
Certain areas in the chromosomes
have repeated DNA sequences. The
number of these sequences varies
from one individual to the next.
• Cutting with restriction enzymes will
produce different sized fragments
depending on the number of repeats.
•
3. Hybridization
Used to detect a particular DNA
segment from multiple bands within a
gel
• In Southern blotting, DNA bands can
be transferred from a gel to a nylon
membrane and then exposed to a
probe.
• Specific probes will base pair to
the DNA if interest
(=hybridization)
D. DNA sequencing
The technique used for reading the
sequence of a DNA molecule
The Sanger Method
Sequencing on DNA microarrays
E. Applications of Recombinant DNA
technology
• Transgenic organisms
• Carry foreign genes
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Gene therapy
• Replacement of a nonfunctioning
gene in somatic cells in an attempt
to fix an inborn genetic error.
Monitoring gene expression
• DNA chips can be used to monitor
gene expression in different cell
types or under different
conditions.
F. Gene Silencing
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Blocking the expression of a
particular gene to look for missing
functions that correspond to that
gene.
Antisense technology
• blocks mRNA which prevents
protein synthesis
Knockout technology
• uses homologous recombination to
swap a disabled form of a gene for
a natural form of the gene