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Chapter 20
DNA Technology and
Genomics
DNA Cloning
Restriction Enzymes
Restriction Enzymes are found in
bacterial cells.
When they cut DNA at a specific
place, they leave sticky ends.
Cloning
 The
cloning vector is the original
plasmid.
After insertion of the gene of
interest into the plasmid the
plasmid is called recombinant
DNA.

Identifying the gene of interest
Usewould
different
How
youprobes
identify different genes?
DNA Libraries



Shotgun approach makes thousands of different
recombinant plasmids
This set of plasmid clones is called a
Besides using plasmids scientists use
as cloning vectors
cDNA Library
Different libraries have
different uses

1.
Which library would you use in the
following scenarios?
You want to clone a gene, without
knowing where and how it is expressed.
genomic library
2.
You want to know regulatory sequences.
genomic library
3.
You want to know the coding sequences
of a gene.
cDNA
library
Expressing Eukaryotic Genes





How can scientists get bacterial cells to
express eukaryotic genes?
Insert the desired gene with a prokaryotic
promoter attached – this is called an
expression vector
How do they remove introns?
Insert cDNA in the first place.
YACs can also be used since they are originally
from eukaryotic cells.
DNA Amplification
 PCR = polymerase chain reaction
 Can make billions of copies of a
target segment of DNA in a few
hours
 can use scanty or impure sources
of DNA (Ex. the blood at the O.J.
Simpson crime scene)

can be used to identify bacteria,
viruses, and genetic sequences for
diseases
Thermocycler
for PCR
Polymerase Chain Reaction (PCR)
Gel Electrophoresis
RFLP - made from noncoding
DNA resulting from differences in the
restriction sites on homologous
chromosomes
 Used
to determine where genes
are located and where they are
expressed
 RFLPs are found on noncoding
segments of DNA and are due to
VNTRs.
Homologous segments of DNA
from a family in which some
members have a genetic disease
Figure 20.x1a Laboratory worker reviewing DNA band pattern
Figure 20.x1b DNA study in CDC laboratory
Southern Blotting Technique
used to detect specific DNA sequences
Restriction fragment analysis by Southern blotting
Entire genomes can be mapped
at the DNA level
•Human Genome Project started
in 1990
Rough Draft – 2000; completed 2003
•James Watson – original director
•Francis Collins – director when it
was finished; now at NIH
•J. Craig Venter – Celera Genomics
Francis Collins, Former Director of
the Human
Genome Project
Ordering of
genetic
markers such
as RFLPs
Mapping the
Genome
ordering of large
overlapping
fragments cloned
in YAC or BAC
vectors
Shotgun
Approach
used by J.
Craig
Venter and
Celera
Genomics
Sanger
Sequencing
Identifying Protein Coding Genes
What would you look for if you
wanted to find an unknown protein
coding gene?
 Scientists use computers to search
for short coding sequences similar
to those present in known genes.
these are called “express service
tags”

Genome Sizes
Genomes
 If there is no pattern between
complexity and number of
genes what makes organisms
more complex?
 More involved regulation
 Alternative splicing of RNA
Determining Gene Function
How can we determine the function of
different genes?
 RNAi; silence a gene and see what it did
 Insert synthetic double stranded RNA’s
that match a gene that will inactivate
translation
 This was used to identify the function of
C. elegans genes

Genome
Wide
Expression
Resulting colors
shows which
genes were being
expressed in each
tissue
Uses two tissue
samples with
differently
colored
fluorescent
nucleotides
What is a DNA Microarray?
•allows scientists to perform an
experiment on thousands of genes
at the same time
•Each spot on a microarray
contains multiple identical strands
of DNA
•each DNA sequence is unique
•each spot represents one gene
Comparing Genomes
Using Microarrays
Has revealed many similar regions even
between yeast and us.
 Can be used to derive evolutionary
relatedness.
 Can use differences between closely
related species to explain phenotypic
differences
 Has led to proteomics (study of full
protein sets)

DNA microarray assay for gene
expression
Practical Applications
Medical
 Disease diagnosis
 Gene Therapy
 Medicines
 Forensics
 Environmental Cleanup
 Agriculture
 Pharm animals
 GMO foods/plants

Pharm Animals
Gene
Therapy
Making
Transgenic
Plants
“Golden” rice contrasted with
ordinary rice
1)The process of separating
DNA fragments to analyze them
is called. ..
2) Why does the DNA migrate
in the gel box?
3) Name two other uses of DNA
fingerprinting that are not related
to crime scenes.
4) The different DNA fragments
that are formed by a fingerprint
are called: