Download stranded DNA from genomic library

Ch 12
DNA Technology and Genomics
DNA Technology
• Methods for studying and manipulating
genetic material
• Cloning
• Genetically Modified
• Gene Therapy
• DNA Profiling
• Genomics
Recombinant DNA
• Combining nucleotide sequences from 2
sources to form a single DNA
• Bacteria often used
– Plasmid
Creating Recombinant DNA
• Restriction Enzymes
– Restirction site
• Sticky Ends
• Ligase
5
Cloning the Gene
• Bacteria containing the gene are cloned
– Typically, antibiotic resistance genes are also
inserted
– Bacteria are grown on antibiotic medium
• All bacteria without the resistance (and target)
gene die
• Only those with gene reproduce
• Stored in genomic library
– Plasmids used to “store” genetic information
Uses
• Gene Cloning
– Produces multiple copies of gene-carrying
DNA
• Genetic Engineering
– Direct manipulation of genes
cDNA
• Express Eukaryotic genes in Prokaryotic
cells
– What about introns?
– Prokaryotes don’t have machinery to splice
• mRNA
– Already spliced
• Work from mRNA to create cDNA
Fig. 12-4
Cell nucleus
DNA of
eukaryotic
gene
Exon Intron
Exon
Intron Exon
1 Transcription
RNA
transcript
2 RNA splicing
mRNA
3 Isolation of mRNA
Test tube
Reverse transcriptase
cDNA strand
being synthesized
and addition of reverse
transcriptase; synthesis
of DNA strand
4 Breakdown of RNA
5 Synthesis of second
DNA strand
cDNA of gene
(no introns)
Uses
• Diagnosis and treatment of disease
• Vaccines
• Therapeutic hormones
Probes
• Genomic Library
– How to find the right gene?
• Nucleic Acid Probe
– Locate specific gene or nucleotide sequence
Probes
• Synthesize short sequence of singe
stranded DNA of complimentary sequence
• Label radioactive tag
• Mix with genomic library
– Bacteria with gene of interest will glow
Fig. 12-5
Radioactive
DNA probe
Mix with singlestranded DNA from
genomic library
Single-stranded
DNA
Base pairing
indicates the
gene of interest
Genetically Modified
• Organisms who have acquired genes by
artificial means
– If from another species= transgenic organism
Agrobacterium tumefaciens
Plant cell
DNA containing
gene for desired trait
1
Ti
plasmid
Insertion of gene
into plasmid
Recombinant
Ti plasmid
3
2
Introduction
into plant
cells
Regeneration
of plant
DNA carrying new gene
Restriction site
Plant with new trait
Gene Therapy
• Altering an afflicted persons genes for
therapeutic purposes
– Treatment of genetically based disorders
– Technology still in infancy
Fig. 12-10
Cloned gene
(normal allele)
1
Insert normal gene
into virus
Viral nucleic acid
Retrovirus
2
Infect bone marrow
cell with virus
3
Viral DNA inserts
into chromosome
Bone marrow
cell from patient
Bone
marrow
4
Inject cells
into patient
DNA Profiling
• Forensics
– Scientific analysis of evidence
• DNA Profiling
– Analysis of DNA fragments
– Determine if they originate form a particular
individual
– PCR
– Gel Electrophoresis
PCR
• 3 step cycle
– Heat separates strands
– Cooling allows primers to form H bonds with
end of target sequence
– DNA Polymerase adds nucleotides
• Creates double stranded DNA
– Repeat
PCR
• Significant breakthrough in genetics
• Original polymerase was from E. coli
– Heating traditionally denatured polymerase
– New polymerase added after each heating
cycle
– Giant pain in the butt
PCR
• Discovery of T. aquaticus
– Lives in hotsprings
– Not denatured under high temps
• Could now be done at higher temps
– Better success rates
PCR
Cycle 1
yields 2 molecules
Genomic
DNA
3
1
3
5
3
Target
sequence
5
5
5
3
Cycle 2
yields 4 molecules
5
5
2 Cool to allow
3
Heat to
primers to form
separate
DNA strands hydrogen bonds
with ends of
target sequences
5
3
5
3
Primer
3
5
DNA
polymerase adds
nucleotides
to the 3 end
of each primer
5
3
New DNA
Cycle 3
yields 8 molecules
Gel Electrophoresis
• Use of gel to separate DNA strands by
size (molecular weight) or charge
• DNA must first be “digested”
– Strands must be cut into different sizes
• Use Restriction Enzymes
– Cut DNA in specific places
• Looks for specific nucletoide sequences
Gel Electrophoresis
Mixture of DNA
molecules of
different sizes
–
–
Longer
molecules
Power
source
Gel
+
Shorter
molecules
+
Figure 12.10
Completed gel
Gel Electrophoresis
• STR
– Short sequences of DNA repeated many
times in a row
– STR analysis compared lengths of STR
sequences at specific sites on the genome
• Create a genetic profile of individuals
– STR’s differ among individuals
Gel Electrophoresis
• RFLP
– Restriction fragment length polymorphisms
– Difference between two samples of
homologous DNA arising from differing
locations of restriction sites
Crime scene
Suspect
w
Cut
C
G
C
G
G
C
G
C
z
A
T
C
G
G
C
G
C
C
G
C
G
G
C
G
C
x
C
G
Cut
C
G
G
C
y
Cut
y
G
C
DNA from chromosomes
– After digestion by restriction enzymes the
fragments are run through a gel
1
–
2
Longer
fragments
z
x
w
Shorter
fragments
Figure 12.11B
+
y
y
• DNA profiling
Crime scene
1 DNA isolated
2 DNA of selected
markers amplified
3 Amplified DNA
compared
Suspect 1
Suspect 2
• Used in forensic investigations
STR site 1
STR site 2
Crime scene DNA
Number of short tandem Number of short tandem
repeats match
repeats do not match
Suspect’s DNA
Genomics
• Studying the entire genome and their
interactions
Human Genome Project
• identify all the approximately 20,000-25,000
genes in human DNA,
• determine the sequences of the 3 billion
chemical base pairs that make up human DNA,
• store this information in databases,
• improve tools for data analysis,
• transfer related technologies to the private
sector, and
• address the ethical, legal, and social issues
(ELSI) that may arise from the project.
Fig. 12-18
Exons (regions of genes coding for protein
or giving rise to rRNA or tRNA) (1.5%)
Repetitive
DNA that
includes
transposable
elements
and related
sequences
(44%)
Introns and
regulatory
sequences
(24%)
Unique
noncoding
DNA (15%)
Repetitive
DNA
unrelated to
transposable
elements
(15%)
HGP
• 3.2 billion nucleotide pairs
– ~21000 genes
– How are we so complex?
• RNA splicing
• Non-coding
• Repetitive DNA
– Telomeres
• Transposable elements
Shotgun Method
1. Chop up genome
1. Squeeze it through a small, pressurized
syringe
2. Clone fragments
1. Insert them into a vector
3. Sequence
4. Search for overlapping segments
5. Reassemble the overlaps
Chromosome
Chop up with
restriction enzyme
DNA fragments
Sequence
fragments
Align
fragments
Reassemble
full sequence
• Recombo DNA
– http://www.youtube.com/watch?v=-sI5vycD2g&feature=related
• PCR
– http://www.youtube.com/watch?v=2KoLnIwoZ
KU&feature=related
– PCR and Gel
• http://www.youtube.com/watch?v=_uoPkavtXgs
Videos
• Ch Overview
– http://www.youtube.com/watch?v=Nc3jArZXHj
s