Download chapter 20: dna technology and genomics

CHAPTER 20: DNA TECHNOLOGY AND GENOMICS
I.
II.
INTRODUCTION
A.
Recombinant DNA
1.
Definition:
B.
Genetic Engineering
1.
Definition:
C.
Biotechnology
1.
Definition:
DNA CLONING
A.
Introduction
1.
Why is it so difficult to work with the just a gene?
B.
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DNA technology makes it possible to clone genes for basic research and
commercial applications: an overview.
1.
Be able to describe the role of plasmids in the cloning of genes
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2.
C.
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Potential uses of cloned genes
Restriction enzymes are used to make recombinant DNA
1.
Restriction Enzymes or Endonucleases
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D.
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2.
Restriction Sites and Restriction fragments
3.
Sticky Ends
Genes can be cloned in recombinant DNA vectors: a closer look
1.
Cloning Vector
a)
This is simply the tool that will carry the gene of interest.
b)
It is usually DNA that will carry the new or foreign gene
into whatever cell we want the gene to be expressed.
c)
Plasmids can be vectors
d)
Bacteria are common host cells that will accept the
plasmid, reproduce the plasmid, and therefore it has cloned
the gene.
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2.
Procedure for Cloning a Eukaryotic Gene in a Bacterial Plasmid
a)
Isolation of vector and gene-source DNA
b)
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Insertion of DNA into the vector
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c)
Introduction of the cloning vector into cells
d)
Cloning of cells (and foreign genes)
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e)
3.
Identification of cell clones carrying the gene of interest
(i)
We can detect the gene or DNA by exposing the
double stranded DNA to chemicals or heat to
denature the DNA and then expose the single
stranded DNA to a radioactive probe of DNA that
pairs up with the gene of interest (Nucleic Acid
Hybridization)
(ii)
Sometimes this probe can have a fluorescent tag or
marker on it, like the GFP that we have spoken
about or will speak about.
(iii)
So what we have done then is to “screen” for the
gene of interest using a nucleic acid probe.
(iv)
Once the bacteria that have the gene of interest we
will want to grow them in large quantities to isolate
the gene product- the protein.
Cloning and Expressing Eukaryotic Genes: Problems and
Solutions
a) Expression Vector: used to overcome some of the difficulties
of putting a eukaryotic gene into a prokaryote.
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b) Presence of noncoding regions
c) YACs or how to avoid those prokaryotic problems
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d) Protein Modifications cannot be done by bacteria
e) Eukaryotic cells just cannot take up the DNA like bacteria so
we Electroporate them!!
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E.
E.
Cloned Genes are stored in DNA libraries
1.
Genomic Library: when a bunch of cut up DNA is exposed to
bacteria, all of the pieces can be taken up by the bacterial plasmids.
So then each plasmid has a particular segment of the DNA that was
all cut up. This is a library of the DNA. Some of these plasmids
will be valuable, others won’t.
2.
Viruses can also be used to make a library: pieces of foreign DNA
can be inserted into the virus’s genome using a restriction enzyme
and ligase. This then is packaged in a capsid and allowed to infect
cells. So as the virus’ DNA replicates, so does the foreign DNA of
interest.
3.
cDNA or complementary DNA (sometimes called copy DNA)
a)
You take all the mRNA that is produced by a cell and
expose it to reverse transcriptase to make DNA from all the
expressed genes. This then is a cDNA Library but it
represents only those genes expressed at the time of the
study.
b)
Liver cells may produce a totally different cDNA library
than lung cells. That way we know what different genes
are being transcribed by these different types of tissue.
The polymerase chain reaction (PCR) clones DNA entirely in vitro
(know this; this was part of one of the essays on the AP Bio Exam 2002)
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III.
DNA ANALYSIS AND GENOMICS
A.
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Introduction
1.
What are some of the questions that can be asked (by Dr. Campbell
as well as the AP Committee) once we have large numbers of
identical segments of DNA?
2.
What is Genomics?
4.
The “other” method to compare samples of DNA –Gel
Electrophoresis.
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B.
Restriction fragment analysis detects DNA differences that affect
restriction sites.
1.
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Be able to describe this method for an essay question. More detail
should come from a lab we do. You are responsible for a detailed
description of this process. Figure 20.8 and 20.9
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2.
Nucleic Acid Hybridization (pg 385 and Fig 20.10)
a) Know the procedure and understand RFLPs( RFLPs was part
of the 2002 AP Exam).
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C.
Entire Genomes can be mapped at the DNA Level
1.
Introduction and the Human Genome Project
2.
Genetic (Linkage) Mapping
a) Genetic mapping is finding out where “genes” are but what we
many times are locating are markers for these genes.
b) When we know the location of various genes we can
determine if they are linked based on their distances between
each other.
3.
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Physical Mapping: Ordering DNA Fragments
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4.
DNA Sequencing
5.
Alternative Approaches to Whole-Genome Sequencing
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D.
Genome sequences provide clues to important biological questions
1.
Introduction
2.
Analyzing DNA Sequences
a)
b)
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Introduction
(i)
Stored in data banks
(ii)
Researchers can scan these sequences to
(iii)
see if a particular gene is similar between organisms
(iv)
promoters are similar
(v)
splicing sites are similar
(vi)
Sequences that are found to be similar are called
Expressed Sequence Tags (ESTs)
Surprisingly Few Genes in the Human Genome
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c)
Studying and Comparing Genes
3.
Studying Gene Expression
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4.
Determining Gene Function
a)
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Disable the gene or mutate it and see what effect it has on
the organism.
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5.
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b)
In vitro mutagenesis: In vitro means to be done outside of
the cell or organism so here a gene is removed so it is
outside of the cell and is altered. The altered form is then
reinserted into the cell and its expression is observed.
Perhaps a protein is deformed or a certain tissue doesn’t
form in an embryo so this suggests the gene has something
to do with controlling cell differentiation.
c)
RNA interference (RNAi): well, I’ve never heard of this
before so. . .
(i)
Purpose: used to shut off the expression of a
specific gene.
(ii)
Take some dsRNA. This dsRNA has a sequence
that matches a gene that will cause the breakdown
of the mRNA that belongs to the gene you are
interested in.
(iii)
So you have no polypeptide made from the mRNA,
hence no function and you can observe the effects.
Future Directions in Genomics
a)
Proteomics
b)
Bioinformatics
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IV.
Practical Applications of DNA Technology
A.
DNA technology is reshaping medicine and the pharmaceutical industry
1.
Introduction
a)
identifying genes of disease and health is part of DNA Tech
b)
finding mutations is part of DNA Tech
c)
understanding how genes are expressed and their control
mechanisms helps you to understand how disease occurs.
d)
Different arrangement of gene expression (microarrays)
help you to understand a larger picture of many genes and
their expression related to disease and health
2.
Diagnosis of Diseases
3.
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Human Gene Therapy
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4.
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Pharmaceutical Products
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B.
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DNA technology offers forensic, environmental, and agricultural
applications
1.
Forensic Uses of DNA Technology
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2.
Environmental Uses of DNA Technology
a)
Bacteria decomposing oil
b)
Bacteria extracting heavy metals from contaminated soil
c)
Bacteria degrade organic pollutants
3.
Agricultural Uses of DNA Technology
a)
Animal Husbandry and “Pharm” Animals
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b)
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Genetic Engineering in Plants
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C.
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DNA technology raises important safety and ethical questions
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