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