Transcript DNA Libraries - Western Washington University

Genetics in the News
Recombinant DNA Technology
…artificial manipulation of DNA.
Recombinant DNA Technology
– isolate DNA,
– cut DNA into workable sized fragments,
– amplify the fragments for storage and subsequent
analysis,
– identify and isolate specific sequences,
– characterize by size genomic location and sequence.
Cutting the DNA
• ~3,100,000,000 base pairs per human
haploid genome,
– average chromosome length is ~13 million base
pairs,
• DNA is cut with bacteria-derived enzymes,
• The act of cutting is termed “digestion”.
Restriction Enzymes
…proteins that recognize specific, short
nucleotide sequences and cut DNA at those
sites,
…bacteria contain over 400 such enzymes
and recognize and cut over 100 DNA
sequences.
Bacteria Defense Mechanism
• restriction enzymes are expressed in
bacteria in order to protect against invasion
by viruses,
• bacterial protect their own DNA by
chemically modifying recognition sites.
Palindromes
stir grits
no lemon, no melon
5’-------G-A-A-T-T-C--------3’
’
’
3 -------C-T-T-A-A-G--------5
EcoRI
’
5 -------G
3’-------C-T-T-A-A
A-A-T-T-C--------3
’
G--------5’
Sticky Ends
...single-stranded DNA overhangs resulting
from restriction digestion.
EcoRI
’
5 -------G
3’-------C-T-T-A-A
A-A-T-T-C--------3
’
G--------5’
RsaI
Rhodopseuomonas sphaeroides
’
’
5 -------G-T
A-C--------3
3’-------C-A
T-G--------5’
Restriction Fragment Length Sizes
(predicted)
- IF 25% A
25% T
25% G
25% C
and
Random Distribution of Nucleotides
i.e. p(specific base) = .25
- THEN Distance between cut sites is equal to 4n bases,
(n = number of base pairs in the recognition site)
Average Restriction Fragment Length
n = 4, 256 base pairs
n = 6, 4096 base pairs
n = 8, 65.5 kb base pairs
Still Lots of Fragments
4 cutter: 3,000,000,000 bp
~256 bp
= 12,000,000 fragments
6 cutter: 3,000,000,000 bp
~4096 bp
= 700,000 fragments
8 cutter: 3,000,000,000 bp
~65.5 kb
= 46,000 fragments
Ligation
…sticky ends with complementary base pairs
can form hydrogen bonds,
…DNA ligase: an enzyme that catalyzes the
reformation of the phosphodiester bonds.
Ligation
5’-------G
’
3 -------C-T-T-A-A
A-A-T-T-C--------3’
G--------5
hydrogen bonds align,
DNA ligase covalently links
’
DNA is Profligate
…sticky ends with complementary base pairs
can form hydrogen bonds,
…with DNA from any species.
Cloning
…specialized DNA technology to produce multiple,
exact copies of a single gene or other segment of DNA
to obtain enough material for further study.
Cloning
…specialized DNA technology to produce multiple,
exact copies of a single gene or other segment of DNA
to obtain enough material for further study.
Requires a Vector…
...a specialized DNA sequence that can enter a living cell,
...signal its presence to an investigator,
... and provide a means of replication for itself and the foreign
DNA it carries.
Mike
And, Vectors…
…contain unique restriction
sites to facilitate the
creation of recombinant
DNA molecules,
... must also possess a
distinguishing physical
characteristic such as size
or shape by which it can be
purified away from the
host cells genome.
Vectors
Plasmid
E. coli
up to 15 kb,
Phage
E. coli
up to 25 kb,
Cosmid
E. coli
up to 45 kb,
BAC
E. coli
100-500 kb,
YAC
Yeast250-1000 kb.
Step 1…restriction digests and ligation of fragments
into cloning vectors,
Cloning Step 2
…vector-insert recombinants are inserted in
host cells,
– Transformation (via bacterial mechanisms);
• plasmids
• BACS
• YACS
– Transduction (via virus mechanisms);
• phage
• cosmids
Cloning Step 3
...vectors contain selectable
markers,
– only cells that contain
vector DNA will survive
selection,
– recombinant vectors can
be discerned from empty
vectors by additional
markers.
Blue/White Cloning
Amplification
…if you were to grind me up with a giant
mortar and pestle, and extract all of my
DNA coding for a single gene, you’d get
about 1 mg of DNA,
…two liters of bacterial culture carrying my
hemoglobin in a recombinant DNA vector,
would yield an equivalent mass of DNA.
Assignment
• Read 10.1 and be prepared to clone a
fragment of DNA using a plasmid as a
vector.
B-PCR vs A-PCR
PCR, Polymeras Chain Reaction
Today
More DNA Science
• DNA Amplification II,
– Polymerase Chain Reaction,
– 6.7, pp. 237-239,
• Gel Electrophoresis,
– Southern Blots, Northern Blots,
– 6.6, pp. 237-238,
• Clones and Libraries, pp
Polymerase Chain Reaction
…invented by Kary Mullis
while cruising in a Honda
Civic on Highway 128
from San Francisco to
Mendocino,
"It was quiet and something
just went, Click!"
Kary B. Mullis
Nobel Laureate, 1993
Chemistry
"THE SUN HAD been hot that day in Mendocino County.
A dry wind had come out of the east, and nobody knew
how hot it had been until, around sunset, the wind
stopped. I drove up from Berkeley through Cloverdale
headed to Anderson Valley. The California buckeyes
poked heavy blossoms out into Highway 128. The pink
and white stalks hanging down into my headlights
looked cold, but they were loaded with warmed oils
that dominated the dimension of smell.
It seemed to be the night of the buckeyes, but something
else was stirring.""My little silver Honda's front tires
pulled us through the mountains. My hands felt the
road and the turns. My mind drifted back to the lab.
DNA chains coiled and floated. Lurid blue and pink
images of electric molecules injected themselves
somewhere between the mountain road and my eyes."
Opening words, Dancing Naked in the Mind Field, © 1998, by Dr. Kary Mullis, Pantheon Books.
Mullis…
... “PCR is a chemical procedure
that will make the structures of
the molecules of our genes as
easy to see as billboards in the
desert and as easy to manipulate
as Tinkertoys”.
DNA
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Making DNA: Components
Cell
PCR
ss DNA
template
dNTPs
helicase, etc.
?
present
present
Primer
primase
?
DNA
Polymerase
DNA
polIII
nucleus
?
Environment
test tube
Oligonucleotides
specific primers
...short pieces of synthetic DNA can
be manufactured that contain any
sequence,
…template specific!
~ Odds of a
Specific Sequence
20-mer: 9.1 x 10-13
Making One Strand Of DNA
add primer
Add Polymerase
Add dNTPs, etc.
Making Two More Strands
Must Denature
Separate Strands
Denaturing
can’t use helicase in vitro
…DNA denaturing conditions such as high
heat or low salt concentrations irreversibly
denature or inactivate most polymerases,
…dNTPs are not affected by denaturation,
…primers are not affected by denaturation.
Making Two More Strands
add primer to second strand
Add polymerase, etc.
Denaturation Step Bad
…several rounds of in vitro replication could
be performed (prior to PCR), however,
accumulation of denatured polymerases
quickly poisoned the reactions.
…bacteria discovered in a hot spring in Yellowstone Natural Park in
1965,
…lives in salty water that ranges from 70o - 75o C,
…thus, does DNA replication at very high temperatures.
Thermus aquaticus’ Enzymes
…basic research demonstrated that many
enzymes isolated from Thermus aquaticus
function at very high temperatures,
…temperatures nearing 100o C,
…DNA denaturating temperatures.
Click
…Kary Mullis realized that repetitive
rounds of DNA synthesis could be
performed by using a heat-stable
polymerase,
… Thermus aquaticus: Taq polymerase.
5’--GCATGCATTAT
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’
3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
CTGATCGTGAC--5’
94
Synthesis
~30 seconds/kb
72o
o
Denature Step
~30 seconds
PCR
~60o
Annealing Step
~30 seconds
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
CTGATCGTGAC--5’
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
CTGATCGTGAC--5’
5’--GCATGCATTAT
3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
5’--GCATGCATTAT
3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
Exponential Synthesis
• as few as 1 DNA templates required,
• excess dNTPS,
• excess primers,
• multiple cycles.
Gel Electrophoresis
…electrophoresis is the movement of
charged particles in an electric
field,
…DNA with it’s phosphate backbone
carries a net negative charge,
– DNA migrates toward the positive
pole in an electrical field.
Size Resolution
…DNA movement during electophoresis is
dependent on several variables,
– strength of the electrical field,
– composition of the matrix (gel),
– charge per unit volume of the molecule,
• each nucleotide has roughly equal charge,
• all DNA molecules have the same charge density,
– size.
Agarose Gel
Agarose: a natural polysaccharide derived
from agar, a substance found in some algae.
Add DNA to Wells, Apply Charge
DNA moves through gel at a rate roughly equivalent
to the inverse log of the number of base pairs.
Visualizing DNA
Ethidium Bromide fluoresces in UV light.
Looks Like This
PCR Applications
…new applications are created every day,
• PCR products can be used for mapping genes,
• PCR products can be used as probes,
• PCR products can be probed,
• PCR can be used to identify genotypes,
• PCR can be used to sequence DNA directly.
Genetics
…in the news.
…a hardcopy of the assigned
paper will earn you two of the
12.5 points on the quiz.
Syllabus Update
• 6.1, 6.6 - 6.8, 10.1 for Wednesday,
– read the rest of the chapter for review,
– Master the Chapter 6 summary,
– Master 6.8, 6.13, 6.18, 6.19, 10.6, 10.7, 10.9, 10.11,
– Quiz through Chapter 6, 7.1 - 7.5, 10.1 Wednesday.
• Read the Science Paper by Friday.
Molecular Probing
Heterologous Hybridization
…genes (DNA), or gene products (RNA) can be
identified based on hybridization to labeled
molecules,
…DNA probes are short, single-stranded stretches of
nucleic acid that are complementary to target nucleic
acids,
– 10 - 1000s of base pairs in length,
…radioactive or fluorescent labeled for detection.
Probe
Add a “labelled” dNTP to an in vitro synthesis reaction.
Southern Blot
Northern Blot
mRNA is...
RNA
DNA Libraries
…collections of cloned DNA fragments,
– genomic,
– cDNA (coding sequences).
Genomic Sequences and Coverage
N = ln(1 - P)
ln(1 - f)
N = number of clones
P = probability of recovering a sequence,
f = fraction of the genome of each clone
Genomic Sequences and Coverage
N=
ln(1 - .99)
ln(1 - v/2,900,000,000)
v = average vector insert size
plasmid (5000 bp) = 2.7 x 106
phage (20 kb) = 6.7 x 105
BAC (125 kb) = 1.0 x 105
YAC (500 kb) = 27,000 clones
E. coli vs. Humans
ln(1 - P)
# Clones = ln(1
- v/g)
P = probability of including any one sequence.
v/g = insert size / genome size
E. coli
Genome = 4.6 mb
n = 4.6 mb / 20 kb insert = 230
P = 0.999
Human
Genome = 2900 mb
n = 2900 mb / 20 kb insert = 145,000
P = 0.999
# Clones = 1585
# Clones = 1,001,621
cDNA
…DNA synthesized from an mRNA template
with the enzyme reverse transcriptase.
Reverse Transcriptase
1. RNA dependent, DNA synthesis.
2. RNA Degradation.
3. DNA dependent, DNA Synthesis.
Error Rate: 1 in 20,000 nucleotides.
mRNA
- polyadenylation.
- introns are spliced out.
-AAAA...
cDNA Construction
in vitro
cDNA
?
Genomic vs cDNA
...Jeff’s hemoglobin…
• isolate red blood cells,
– red blood cells make lots of hemoglobin, thus the
mRNA is enriched for hemoglobin sequences,
• construct a cDNA library,
• isolation of hemoglobin clones is facilitated,
– genomic: ~1 of 1,000,000 clones,
– cDNA from red blood cells: 1 of 3 clones.
cDNA Libraries
…provide a ‘snap-shot’ of the genes
expressed in a particular cell, at a particular
time, or under specific condition,
…however, do not provide regulatory
sequences.
Assignment
• Study figure 6.27, pp 237,
• Be able to describe the steps required to
isolate a genomic clone using a cDNA clone
as a molecular probe.
Primers?
QuickTime™ and a
Cinepak decompressor
are needed to see this picture.
Cycle Sequencing
PCR: 1 Strand Sequencing
• PCR driven DNA sequence procedure,
– non-exponential amplification,
• Dideoxy sequencing method,
– florescent indicators.
Single Strand PCR
dNTPs
Template
1 Primer
=
Taq Polymerase w/ Buffer
Cycles
Polymerization until Taq falls off, linear amplification.
Cycle Sequencing
Chain Termination
ddNTPs
dNTPs
Template
1 Primer
=
Taq Polymerase w/ Buffer
Cycles
Polymerization until Taq hits ddNTP, Taq falls off.
Fluorescent ddNTPs
Plus: a preponderance of dNTPs
Cycle Sequencing
Chain Termination
ddNTPs
dNTPs
Template
Another
Template
etc.
Lots of each sized fragment are produced, each with a
specific florescent base on the end.base
For Friday!
…READ IT! BRING YOUR COPY!
Exam #1
A range: | | | | | | |
B range: | | | | | | |
C range: | | | | | | | | | | | |
D range: | | | | | | | | | | |
Failing: | | | | | | | | | | | | |
Mean = 72%
Median = 72%
Mode = 76%
year
Quiz 1 Quiz 2 Quiz 3 Quiz 4 Exam 1 Exam 2
2007 A 11.5
2006 S 12
2007 S 11.3
8
8
9.2
9
10.7
11
7
10
11
72%
78%
79%
66%
67%
Increase your study time, or see me to work on study
approaches.
Albinism is a recessive trait
in humans.
Assignment: figure out this pedigree. 14/50