Download Cloning and Sequencing

Cloning and
Sequencing
Project overview
Background
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Project will have you cloning the gene that codes
for the enzyme glyceraldehyde-3-phosphate
dehydrogenase (GAPDH)
GAPDH is a housekeeping gene necessary for
survival
GAPDH is an enzyme that is crucial for
glycolysis to occur
Glycolysis
GAPDH
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can be easily isolated in cells
Is made up of four subunits that are either identical
(homotetramer) or in pairs of slightly different proteins
(heterodimer)
Has two domains: amino terminal region binds to
NAD+ while the carboxy terminal region has the
dehydrogenase activity
Does two things:
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Removes H+ from GAP and transfers
it to NAD+
Adds second Phosphate to GAP
GAPDH genes
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Found in the cytosol (glycolysis) and in the
chloroplast as part of photosynthesis
Isozymes coded for on nuclear DNA
GAPC denotes the gene that codes for cytosolic
GAPDH and is the gene that we will study.
The GAPC protein is a heterodimer.
Gene Cloning
Big picture for this unit
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Isolate GAPC gene from plants
Amplify the GAPC gene by nested PCR
Assess the results of the PCR
Purify the PCR product containing GAPC
Ligate (insert) GAPC gene into plasmid vector
Transform bacteria with new plasmid
Isolate plasmid from bacteria
Confirm plasmid by restriction digests
Prepare plasmid DNA to be sequenced by outside
facility
Analyze sequence of your GAPC gene using
bioinformatics
Nucleic Acid Extraction
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Task is to separate DNA from rest of the cellular
components, including membranes, proteins, and
enzymes
Must also remain in tact after extraction
Plant cells also have a cell wall to disrupt
Nucleases can digest DNA
Acidic contents of organelles can damage DNA
Some plants have polyphenols that bind to DNA
rendering it useless for experiments
Basic Steps of DNA Extraction
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Harvest cells from fresh, young plants
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Grind cells to physically disrupt tissue & cell walls
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Lyse cells to disrupt membranes
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Remove cellular debris by centrifugation
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Digest remaining cellular proteins
Basic Steps of DNA Extraction
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Purify DNA by ion-exchange chromatography
to remove contaminants
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Concentrate DNA by ethanol precipitation
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Determine purity and concentration of DNA
with UV Spec
Lysis Buffers
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EDTA to destabilize the membrane and inhibit
nucleases
Buffers to maintain pH since acids are released
by organelles
Detergent to dissolve membrane
DTT denatures proteins
Polymerase Chain Reaction
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Rapidly creates multiple copies of a segment of
DNA
Uses repeated cycles of DNA synthesis in vitro
Used in DNA fingerprinting, kinship analysis,
genetic testing for mutations, and infectious
disease for diagnosis
PCR
Round 0 = 1 copy
Round 35 = billions of copies
PCR players
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DNA template – targeted piece of DNA
Primers – small segments of DNA that bind
complementary upstream and downstream of
the target on the template
Taq DNA polymerase – isolated from the
Thermus aquaticus bacteria found in hotsprings of
Yellowstone Park
DNA nucleotides in the form of
deoxynucleoside triphosphates (dNTPs)
Reaction Buffer – maintains pH for enzymes
General PCR Process
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Denaturation – split apart the two DNA strands
by heating them to 95oC for 1 min
Annealing – primers bind to target sequence by
cooling reaction to 40-60oC for 1 min
Extension – Taq Polymerase extends the primers
and copies each DNA template strand by
heating to 72oC for 1 min
Primers
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Required for both sides of the target sequence (forward
& reverse primer)
Length of primer is generally 18-30 nucleotides
G/C content and intra-complementarity are a concern
when designing primers
Actually not a single primer for each but a mixture of
primers (oligoprimers) if the sequence of the target is
not known
If amino acid sequence of gene product is used then
degenerate primers must be used
Initial forward primer is
GABTATGTTGTTGARTCTTCWGG
B=G/T/C R=G/A (purines) W =A/T
Nested PCR
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Initial PCR primers are degenerate and based on
a consensus sequence
The chances that the initial primers will bind to
sequences other than the target are high
A second set of primers designed to be more
specific to GAPC is used
They are nested within the initial primers and
are not degenerate thus much more specific to
the GAPC gene
Nested PCR
Set-up
Our experiment
Tube 1: negative control (no DNA)
Tube 2: Arabidopsis gDNA
Tube 3: Positive control pGAP plasmid
Tube 4: Your plant DNA
PCR Plan
Initial Denaturation
1st round
2nd round (nested)
95oC for 5 minutes
95oC for 5 minutes
Denaturation
95oC for 1 minute
95oC for 1 minute
Annealing
52oC for 1 minute
46oC for 1 minute
Extention
72oC for 2 minutes
72oC for 2 minutes
Final Extension
72oC for 6 minutes
72oC for 6 minutes
Hold
15oC forever
15oC forever
Then 40 Cycles of:
Gel Electrophoresis
PCR purification
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Small impurities can have a negative effect on
the ligation of the PCR product to vector DNA
Impurities include unincorporated dNTPs,
polymerases, primers and small primer-dimers.
A PCR Kleen spin column will remove the
impurities in less than 4 min.
Gene Cloning
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Cloning is the production of exact copies of a
piece of DNA.
It requires ligating (splicing) the PCR product
into a cloning vector – often a plasmid DNA
The recombinant DNA of the ligation product
can now be put into a cell to propagate
(replicated)
Plasmids are good vectors:
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small (2,000 – 10,000 bp)
circular, self-replicating
high copy number
multiple cloning sites (MCS)
selectable markers (Amp-resistance)
screening (reporter genes, positive select)
control mechanisms (lac operon)
can handle the size of the insert
pJet1.3 blunted vector
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Designed for blunt-end cloning
High copy number
Contains Amp-resistant gene
Contains eco47IR gene which allows for positive
selection
It is 2,974 bp long
Inserts
Sticky ends have single strands of nucleotides
on ends and are good for directional inserting
 Blunt ends have no single
strands and thus are easier
to insert but are non
directional.
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Ligation
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T4 DNA Ligase catalyzes formation of
phosphodiesterase bond between 3’ hydroxy on
one piece and the 5’ phosphate on another
piece.
Requires ATP and Mg+2
Insert to vector DNA ratio should be 1:1
Proofing reading DNA polymerase removes
dangling 3’A of PCR product
Products of Ligation
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Self-ligation of vector
Ligation of vector to primer-dimers
Ligation of multiple inserts
Self-ligation of inserts
Ligation of one insert into vector
Transformation
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Once PCR product (insert) has been ligated into
a plasmid, the plasmid be introduced into a
living bacterial cell to replicate.
Two methods of transformation:
Electroporation
 Heat Shock
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Both methods make cells competent - able to
take up plasmids
Transformation Steps
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Wash away growth media from cells
Place cells in ice cold calcium chloride which most
likely hardens the cell membrane
Add plasmid to cells
Move cells to hot environment (usually 42oC) causes
membrane pores to open so plasmid can enter
Add nutrient media to cells to allow them to recover
from stress
Plate cells on selective growth plates (Amp and IPTG
(increases expression of ampr gene)
Microbial Culturing
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Pick a colony from the transformed cells to
innoculate a liquid culture
Liquid culture (broth) must have selective
antibiotic (Amp) in it.
Choose a single colony from the plate
Under favorable conditions, a single bacteria
divides every 20 minutes and will multiply into
billions in 24 hours
Plasmid Purification
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To confirm that the engineered cells have been
transformed with the correct DNA
Different methods
Lysozyme Method
 Alkaline Cell Lysis Method
 Column Methods (Aurum)
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Plasmid preps
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Spectrophotometer determination of culture
density. Take OD600 of culture (equal to about
8x108 cells/ml
Aurum column can process up to 12 OD●ml of
bacterial host cells
Cells disrupted with a lysis buffer
DNA binds to membrane of column, is washed
and then eluted with aqueous buffer.
Restriction Digests
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DNA cut with restriction enzymes
Evolved by bacteria to protect against viral
DNA infection
Endonucleases -cleave within DNA strands
Over 3000 known enzymes
Restriction Digests
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Each enzyme cuts DNA atEnzyme cuts
a specific sequence=
restriction site
Many of the restriction
sites are 4 or 6-base
palindrome sequences
Fragment 1
Fragment 2
Enzyme Examples
EcoRI
G-A-A-T-T-C
C-T-T-A-A-G
HindIII
A-A-G-C-T-T
T-T-C-G-A-A
BamHI
G-G-A-T-C-C
C-C-T-A-G-G
Bgl II
A-G-A-T-C-T
T-C-T-A-G-A
Restriction Digest
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Restriction Buffer provides optimal conditions:
NaCl provides correct ionic strength
 Tris-HCl provides proper pH
 Mg+2 is an enzyme co-factor
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Body temperature (37oC) is optimal
Too hot kills enzyme
 Too cool takes longer digestion time
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DNA Sequencing
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Determining the exact order of the nucleotide
sequence in a DNA molecule.
Use to take days, now takes hours
Have sequences of entire genones for over 700
organisms
Sanger Method
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Prepare single-stranded DNA template to be sequenced
Divide DNA into four test tubes
Add primer to each tube to start DNA synthesis
Add DNA polymerase
Add labeled deoxynucleotides (dNTP) in excess.
Labeled with radioactive or fluorescent tags
Add a single type of dideoxynucleotides (ddNTPs) to
each tube. When incorporated in sythesized strand,
synthesis terminates.
Allow DNA synthesis to proceed in each tube
Run newly synthesized DNA on a polyacrylamide gel
Reading the Sequence
• In the tube with the ddTTP, every time it is time to
add a T to the new strand, some Ts will be dTTP and
some will be ddTTP.
• When the ddTTP is added, then extension stops and
you have a DNA fragment of a particular length.
• The T tube will, therefore, have a series of DNA
fragments that each terminate with a ddTTP.
• Thus the T tube will show you everywhere there is a
T on the gel
• Same thing happens in all tubes
• Read gel from top to bottom looking at all four lanes
to get the sequence.
Automated Sequencing
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Dye-terminator sequencing labels each of the
ddNTPs with a different color fluorescent dye.
Now reaction can be run in one tube
Use capillary electrophoresis rather than the
standard polyacrylamide slab gel.
When DNA fragment exits gel, the dyes are
excited by a laser and emit a light that can be
detected .
Produces a graph called a chromatogram or
electopherogram
Automated Sequencing
Bioinformatics
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Computerized databases to store, organize, and
index the data and for specialized tools to view
and analyze biological data
Uses include
Evolutionary biology
 Protein modeling
 Genome mapping
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Databases are accessible to the public
Allow us to record, compare, or identify a DNA
sequence