Cloning and
Sequencing
Project overview
Background
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
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:
Removes H+ from GAP and transfers
it to NAD+
Adds second Phosphate to GAP
GAPDH genes
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
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
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
Harvest cells from fresh, young plants
Grind cells to physically disrupt tissue &
cell walls
Lyse cells to disrupt membranes
Remove cellular debris by centrifugation
Digest remaining cellular proteins
Basic Steps of DNA
Extraction
Purify DNA by ion-exchange
chromatography to remove
contaminants
Concentrate DNA by ethanol
precipitation
Determine purity and concentration of
DNA with UV Spec
Lysis Buffers
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
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
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
General PCR Process
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
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
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 1st round 2nd
round (nested)
Initial Denaturation 95oC for 5 minutes 95oC for 5
minutes
Then 40 Cycles of:
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
Gel Electrophoresis
PCR purification
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
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:
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
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.
Ligation
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
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
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
Both methods make cells competent
- able to take up plasmids
Transformation Steps
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
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
To confirm that the engineered cells
have been transformed with the
correct DNA
Different methods
Lysozyme Method
Alkaline Cell Lysis Method
Column Methods (Aurum)
Plasmid preps
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
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
Enzyme cuts
Each enzyme cuts
DNA at 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
Restriction Buffer provides optimal
conditions:
NaCl provides correct ionic strength
Tris-HCl provides proper pH
Mg+2 is an enzyme co-factor
Body temperature (37oC) is optimal
Too hot kills enzyme
Too cool takes longer digestion time
DNA Sequencing
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
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
•
Automated Sequencing
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
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
Databases are accessible to the public
Allow us to record, compare, or
identify a DNA sequence
