Biol 1208r overview Where are we today How does pcr work Perform pcr reactions overview Where are we today Initial seawater inoculations Back up amp Grow positives to larger concentrations ID: 932039
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Slide1
Pcr
MARCH 10, 2015
Lab 7
Biol
1208(r)
Slide2overview
Where are we today?
How does
pcr
work?
Perform
pcr
reactions
Slide3overview
Where are we today?
Initial sea-water inoculations
Back up & Grow positives to larger concentrations
Molecular characterization of isolated organism
Slide4Sea water (1 cell/uL)
5 uL
Distilled water
Inoculation tubes with GOM-like media
3 weeks at 30°C
Flow cytometry
+
+
+
DMSO stock
Flask
2 weeks at 30°C
+
PCR 16S rRNA gene
Identify organism
DNA extraction
Sequence comparison with known organisms
DNA sequencing
-
Slide5overview
Where are we today?
Initial sea-water inoculations
Back up & Grow positives to larger concentrations
Molecular characterization of isolated organism
Slide6Sea water (1 cell/uL)
5 uL
Distilled water
Inoculation tubes with GOM-like media
3 weeks at 30°C
Flow cytometry
+
+
+
DMSO stock
Flask
2 weeks at 30°C
+
PCR 16S rRNA gene
Identify organism
DNA extraction
Sequence comparison with known organisms
DNA sequencing
-
Slide7+
1000 cells
1000 molecules of DNA
Target a portion of the total DNA in each molecule
Make a billion+ copies of just that region!
Read the genetic code in those fragments of DNA
Identify the organism from its genetic code
DNA extraction
PCR
DNA sequencing & analysis
Slide8overview
Where are we today?
Initial sea-water inoculations
Back up & Grow positives to larger concentrations
Molecular characterization of isolated organism
Slide9Sea water (1 cell/uL)
5 uL
Distilled water
Inoculation tubes with GOM-like media
3 weeks at 30°C
Flow cytometry
+
+
+
DMSO stock
Flask
2 weeks at 30°C
+
PCR 16S rRNA gene
Identify organism
DNA extraction
Sequence comparison with known organisms
DNA sequencing
-
Slide10What does pcr stand for?
Political course for runaways
Painstakingly crafting our rafts
Polymerase chain reaction
Slide11Pcr is a way of amplifying all the dna of an organism without any selection process.
True
False
Slide12Pcr is a way of amplifying a targeted region of the genome of an organism.
True
False
Genome
= total genetic material in a cell
Slide13What is PCR?
PCR:
Polymerase Chain Reaction
pcr
is a process by which we can amplify
a targeted portion of the genome
to a billion copies starting with one or only a few copies.
http://www.lifetechnologies.com/us/en/home/life-science/pcr/elevate-pcr-research/pcr-video-library/pcr-animation.html
Slide14PCR:
Polymerase Chain Reaction
Elongation is done by
DNA polymerase
. Needs to be heat-stable to withstand the near-boiling denaturation temperatures!
Taq
polymerase
: isolated from bacteria
Thermus
aquaticus
living in hot geysers!
The key to automating PCR!
Slide15PCR:
Polymerase Chain Reaction
1. Separate the two DNA strands using heat (near-boiling temperatures)
DENATURATION
Slide16PCR:
Polymerase Chain Reaction
Separate the two DNA strands (
denaturation
)
Target the portion of the genome you want to make billions of copies of using “primers”
ANNEALING
Called “annealing” because primers
anneal
(or stick) to the original DNA strand
Slide17PCR:
Polymerase Chain Reaction
Separate the two DNA strands (
denaturation
)
Target the portion of the genome you want to make billions of copies (
annealing
)
Make a new strand of DNA using
Taq
DNA polymerase & the building blocks of DNA (dNTPs)
ELONGATION
(the construction worker)
(the bricks)
Slide18PCR:
Polymerase Chain Reaction
Separate the two DNA strands (
denaturation
)
Target the portion of the genome you want to make billions of copies (
annealing
)
Make a new strand of DNA (
elongation
)Do 1, 2, 3 over and over again to make billions of copies of your targeted segment!
Slide19Choose the correct order for the 3 major steps to successfully perform a pcr.
Denaturation, elongation, termination
Denaturation, annealing, termination
Denaturation, elongation, annealing
Denaturation, annealing, elongation
Slide20What’s in the pcr reaction Tube?
The “major” components:
DNA
Primers
Dna building blocks:
polymerase + magnesium
OTHER COMPONENTS:
clean water
buffer
dNTPs
Taq
Taq
A
T
G
C
Slide21template dna
:
double-stranded
,
containing the segment
that will be copied
several times
Primers
: small segments of single-stranded DNA, bind to a specific region on either side of the target DNA sequence and initiates replication of the target DNA at that point
Primers specify the DNA sequence to be amplified PROVIDE a starting point for taq polymerase to start adding
What’s in the pcr reaction Tube?
dNTPs
Slide22: deoxyribonucleotides – building blocks of new DNA strand
Taq Polymerase
:
an enzyme that makes a new strand of DNA through the sequential addition of nucleotides (requires
magnesium
for its activity)
Why is
taq polymerase so special?dNTPs
What’s in the pcr reaction Tube?
Taq
A
T
G
C
Slide23Why do we need the “special” taq polymerase for pcr
instead of using
dna
polymerase from humans?
Because
taq
polymerase can withstand the near-boiling temperature used for denaturation whereas human polymerase cannot
‘
cuz
taq just sounds cool!It is traditional – taq
is just how they always did it!
Slide24Taq polymerase
Key to automating PCR!
Thermus
aquaticus
live in very high temperature conditions in hot geysers. Hence, its enzymes can withstand the near-boiling denaturation temperatures.
Most enzymes from organisms that live in ambient conditions like we do, die under 95-98°C temperatures!
Until Taq polymerase was discovered and developed for use, people had to stop the PCR reaction after each cycle and add new polymerase because these enzymes would have denatured at the high denaturation temperatures!
Slide25Buffer
:
a salt-solution that helps to stabilize the DNA and other components of the reaction.
Water
:
brings the solution “to volume”
KNOW THE FUNCTIONS OF
ALL
THE THINGS THAT GO INTO A PCR REACTION TUBE and what they are!
What’s in the pcr reaction Tube?
Slide26WHICH COMPONENT OF PCR ALLOWS YOU TO AMPLIFY ONLY A TARGETED PORTION OF A GENOME?
Template dna
Primers
Buffer
Deoxyribonucleotides
Taq polymerase
Slide2716S ribosomal rna gene primers
Ribosomes (protein factory of the cell) have a large and small subunit.
The 16S ribosomal RNA (rRNA) is a part of the small ribosomal subunit.
The 16S rRNA gene (rDNA) is the most conserved (least variable) gene amongst prokaryotic organisms.
However, small segments of this gene show variability amongst different organisms; called
hypervariable regions.
The hypervariable regions are
flanked by highly conserved regions
.
Slide28For organism identification purposes, we must compare the ____ against a database:
The conserved regions
The hypervariable regions
Somewhere else from above
Slide29in order to amplify the max number of microbes, the primers should lie on:
The conserved regions
The hypervariable regions
Somewhere else from above
Slide3016S ribosomal rna gene primers
Organism A
Organism B
Organism C
Organism D
conserved
conserved
hypervariable region
Design primers ON the conserved region TO AMPLIFY the hypervariable region
Slide31andy is concerned that he may not have switched out pipet tips between his sample pcr
and the
negative
control.
If what he thinks is correct:
his negative control (
water
) should contain
pcr
product
His negative control (water) should not contain pcr product
His negative control (known bacteria dna) should contain
pcr product
Slide32Sandy did not get a pcr product though she was expecting one. She is wondering if she forgot to include one of the
pcr
ingredients. Luckily she has a
positive
control!
If what she thinks is correct:
her positive control (
KNOWN BACTERIA DNA
) should contain
pcr product
Her positive control (water) should not contain pcr
productHer positive control (known bacteria
dna) should not contain pcr product
Slide33Analysis of pcr fragments
LAB 8
BIOL 1208(R)
March 17, 2016
Slide34How do you know your pcr worked correctly???
You visualize it!!!
When you design primers for a PCR product, you can predict the size of the product.
(Remember: Primers flank the region that you want to amplify by PCR)
Check to see if it’s the right size.
Slide35What should you expect your pcr product to be?
ATGG
CTCGA
TCGTACGTTTCCCGGGATTCGG
GTCAA
GGTCC
Full DNA sequence:
Primer 2
Option A:
CTCGA
TCGTACGTTTCCCGGGATTCGG
GTCAA
Primer 1
Option B:
Option C:
ATGG
CTCGA
TCGTACGTTTCCCGGGATTCGG
GTCAA
GGTCC
TCGTACGTTTCCCGGGATTCGG
Option A
Option B
Option C
32 bp (
b
ase
p
airs)
Slide36Visualizing pcr product on a gel by gel electrophoresis
When you design primers for a PCR product, you can predict the size of the product.
(Remember: Primers flank the region that you want to amplify by PCR)
Check to see if it’s the right size.
ATGG
CTCGA
TCGTACGTTTCCCGGGATTCGG
GTCAA
GGTCC
Full DNA sequence:
Primer 2
PCR product (
32 bp
):
CTCGA
TCGTACGTTTCCCGGGATTCGG
GTCAA
Primer 1
10 bp
20 bp
30 bp
50 bp
32 bp?
Slide37overview
About gel electrophoresis
How can gel electrophoresis be used to see if your PCR worked
Is there a better way, more fool-proof way to confirm your PCR?
Look at the actual sequence of DNA, not just the size
Sanger sequencing
Slide38Gel electrophoresis as a chromatography technique
Slide39Chromatography
Collective term for a set of laboratory techniques used for the
separation of mixtures
(based on various factors depending on the specific technique)
Chromatography involves
2 phases
:
Mobile phase
Stationary phase
(can be different things for different chromatography techniques)
Slide40Gel electrophoresis (in general)
Gel electrophoresis
is one type of chromatography:
for the
separation of macromolecules (DNA, proteins, etc.)
using
electricity
based on
size
and/or charge
Mobile phase:
what moves the molecules Stationary phase: what do the molecules move on (the support)
Slide41What do you think is the mobile phase in gel electrophoresis?
Electricity
Hydrogen & Oxygen (gases)
Gel matrix
Buffer
DNA
Slide42What do you think is the stationary phase in gel electrophoresis?
Electricity
Hydrogen & Oxygen (gases)
Gel matrix
Buffer
DNA
Slide43Gel electrophoresis
Gel electrophoresis
is one type of chromatography:
for the
separation of macromolecules
using
electricity
based on
size and/or charge
Mobile phase:
electricity Stationary phase: gel
matrix
Slide44Example 1: protein gels
Proteins with net (+) charge attracted to the (-) end and vice versa
opposites attract!
Separation is based on
size AND charge
Smaller proteins run farther down/up the gel.
Which is the largest protein? What is its net charge?
Which is the smallest protein? What is its net charge?
Black end
(-)
Red end
(+)
1.
They are less caught up in the gel matrix and “snake through” faster.
2.
As they get closer to the opposite end, the attractive power increases and they start to travel even faster.
1
2
3
4
5
6
7
8
Slide45Example 2: dna gels
DNA is
negatively charged
because of the phosphate backbone and so is only attracted to the positive end.
Separation is based on
size ONLY
.
Smaller DNA fragments run farther down the gel (for the same reasons as before).
Which is the smallest DNA fragment?
Which is the largest DNA fragment?
Black end
(-)
Red end
(+)
1
2
3
4
5
6
7
8
Slide46Why did we put the gels into the electrophoresis rigs with wells at the black end?
Because DNA is negatively charged and runs towards the red (+) end
Because DNA is positively charged and runs towards the red (-) end
Slide47Why is separation of dna only based on size?
It is not; it’s based on size and charge
Because they all have the same charge, so separation is only based on size
Because DNA has no charge, so separation is only based on size
Slide48example dna gel
Once your gel is done running, we will visualize it like this too under the dark reader.
Size marker
Smaller fragments run farther down the gel
Slide49Visualizing dna on the gel
The fluorescing bands are DNA.
Fluorescence is due to a compound called
SYBR-Green
which is in your dye mix.
SYBR-Green:
binds to DNA
DNA-dye complex absorbs
blue light DNA-dye complex emits green light
(fluorescence!)
There are other compounds like SYBR-Green also, e.g. SYBR-Gold, ethidium
bromide.
If asked how SYBR Green works,
these are the 3 points I am looking for!
Slide50Is that band the right (intended) product?
How do you know if you have the right band (
i.e.
if you amplified the right fragment)?
Check to see if fragment is the right (expected) size
Sequence the fragment
Size marker/ladder
Your band should close to HERE
(1465 bp or ~1.5 kb)
DNA fragments of known sizes, used to compare your band against to determine its size
Slide51Dna sequencing
Developed by
Frederick Sanger
in 1977 (Nobel prize, 1980)
Sanger also won the Nobel prize for developing protein sequencing!
Sanger sequencing/Sanger chain termination method
A slight variation of the method is still widely used today
Dideoxynucleotides
(ddNTPs)
Image source: www.nwfsc.noaa.gov
Slide525’ – A T C G C G T A
TTTT
– 3’
AAAA
– 5’
(primer)
Polymerase +
dNTPs
+
ddA
ddT
ddC
ddG
*
*
*
*
A T
AAAA
– 5’
*
A G C G C A T
AAAA
– 5’
*
T
AAAA
– 5’
T A G C G C A T
AAAA
– 5’
*
*
C A T
AAAA
– 5’
C G C A T
AAAA
– 5’
*
*
G C A T
AAAA
– 5’
G C G C A T
AAAA
– 5’
*
*
(6 bp)
(11 bp)
(5 bp)
(12 bp)
(7 bp)
(9 bp)
(8 bp)
(10 bp)
ddA
ddC
ddT
ddG
T
A
C
G
C
G
A
T
3’
5’
5’ – A T C G C G T A
TTTT
– 3’
T A G C G C A T
AAAA
– 5’
3’ –
Slide53Dna sequencing
Developed by
Frederick Sanger
in 1977 (Nobel prize, 1980)
Sanger also won the Nobel prize for developing protein sequencing!
Sanger sequencing/Sanger chain termination method
Current methods
used fluorescent-labeled
ddNTPs
and a
capillary gel/laser + computer to read the bases
https://www.youtube.com/watch?v=e2G5zx-OJIw
Slide54Sequencing results can tell us pure cultures from mixed cultures.
True
False
Slide55Sequencing can identify pure culture vs. mixed culture
Can you tell the pure cultures from the mixed culture?
http://scienceblogs.com/digitalbio/2007/11/15/match-the-trace-with-the-sampl/
Different organisms
different 16S rDNA sequence in the HV region different chromatogram!
Slide56Molecular analysesLAB 9
BIOL 1208(R)
March 31, 2016
Slide57Gel electrophoresis
Sea water (1 cell/uL)
5 uL
Sterile media
Inoculation tubes with GOM-like media
3 weeks at 30°C
Flow cytometry
+
+
+
DMSO stock
Flask
2 weeks at 30°C
+
Sequence analysis
PCR 16S rDNA
Identify organism
DNA extraction
Slide58OVERVIEW
Quick recall: Sanger sequencing
How to analyze sequencing data
Perform analysis on sequencing data
Slide59https://www.youtube.com/watch?v=e2G5zx-OJIw
SANGER SEQUENCING
Sanger sequencing uses ddNTP to terminate the growing DNA chain and thereby determine the position of each base.
Chromatogram peaks can be used to derive the DNA sequence (5’
3’)
Slide60Can you tell the pure cultures from the mixed culture?
http://scienceblogs.com/digitalbio/2007/11/15/match-the-trace-with-the-sampl/
Slide61Sequence analysis:
making contigs
Sanger sequencing can yield
~700-900 bp
of sequence information from both DNA strands.
Check for overlapping sequences to make a “longer consensus sequence” called
contig
.
http://www.gene-quantification.com/wishart-dna-sequencing.pdf
Slide62Sequence comparison using
blast
What is it ACTUALLY doing when you click the button??
Align the sequence you put in against database containing genome sequences for many (many!!!) organisms.
Find possible sequence matches and calculates the divergence,
i.e.
how many mutations are there between your sequence and the reference sequence.
Lowest number of mutations = most closely related.
Slide63Perform blast analyses
Take good notes!!!
http://reverse-complement.com/
http://doua.prabi.fr/cgi-bin/run_cap3
http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&BLAST_PROGRAMS=blastn&PAGETYPE=BlastSearch&DATABASE=refseq_rna&DESCRIPTIONS=100&EQ_TEXT=arabidopsis[orgn]&QUERY=8033
Reverse-Complement
:
Create contig
:
BLAST
(sequence comparisons):
See further details about this works in the BLAST how-to-guide handout