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Techniques in Cloning
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 enzymes
General PCR Process
• Denaturation – split apart the two DNA
strands by heating them to 95oC for 30s -1 min
• Annealing – primers bind to target sequence
by cooling reaction to 40-60oC for 30s - 1 min
• Extension – Taq Polymerase extends the
primers and copies each DNA template strand
by heating to 72oC for 30s - 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 target is used
• They are nested within the initial primers and
are not degenerate thus much more specific
to the target gene
Nested PCR
Our experiment
Tube setup:
Add the following to a P3 tube (with PCR reaction pellet)
5 ul
10 ul
15 ul
Target DNA template (P1)
Primer set (P2)
Enzyme -grade water (P4)
PCR Plan
Initial Denaturation
94oC for 5 minutes
Then 30 Cycles of:
Denaturation
94oC for 30 sec
Annealing
50oC for 30 sec
Extention
72oC for 30 sec
Final Extension
72oC for 5 minutes
Hold
15oC forever
Gel Electrophoresis
• Definition: the process of separating
molecules based on size and charge
• Agarose: highly purified agar, heated and
dissolved in buffer. Forms a matrix of pores
for molecules to travel through.
– Smaller molecules travel further
– Molecules migrate towards the positive (red) end
of the chamber
Gel Electrophoresis
• Process
– Make Agarose gel
• Thinner gels (0.8%) yield better results for larger DNA
– Prepare samples
• Restriction enzymes used to cleave at specified sites
– Apply samples to gels, apply current
• If samples run from positive end they will run off the gel
– Stain gels to see bands
• Would not be able to see bands if we did not stain
Gel Electrophoresis
• DNA molecules have a negative charge
– This allows them to migrate towards the positive end of
the chamber
• The samples and the electrophoresis chamber use
specialized buffers. Using TAE/TBE buffer helps
stabilize the sample and allows the reaction to occur
quicker in the chamber.
– If water were in the chamber instead of TAE/TBE buffer the
reaction would take much longer or migration may not
occur at all
• Stains: ethidium bromide will cause the bands to
glow orange under UV light. Fast stain will result in
blue bands
Uses for Gel Electrophoresis
• DNA fingerprinting or profiling
– Paternity testing
– Crime scene sample analysis
– Identification of bacteria and other pathogens
• Who is credited with discovering the DNA
profiling process?
– Alec Jefferies in 1985
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 primerdimers.
• A PCR spin column will remove the impurities
in less than 4 min.
Restriction enzymes (endonucleases)
• Definition: class of enzymes that cleave (cut) DNA at
a specific and unique internal location along its
length.
– Makes 2 incisions, one through each of the sugar-phoshate
backbones of the double helix
• They can be naturally produced in bacteria and the
bacteria use them as a defense mechanism against
viral infection
– The enzymes chop up the viral nucleic acids and destroy
the virus
– More than 3,000 known restriction enzymes
– Common ones are: EcoRI, Psti, HindII
Restriction enzymes (endonucleases)
• Discovered in late 1970s by Arber, Smith and
Nathans
• The chemical bonds that the enzymes cleave can be
reformed by other enzymes known as ligases
• Uses:
– To insert new segment of DNA
– To cut specific segments of DNA to study
– To cut segment from one gene to insert it into another
• Genetic engineering or recombinant DNA
• Need suitable host, vector for carrying plasmid, way to get host to
take up gene
Restriction Digests
• 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 enzymes (endonucleases)
• Sticky ends: when unpaired length of bases (5’
AATT 3’) encounter an unpaired length of
sequences (3’ TTAA 5’), they will bind or are
“sticky” for each other.
• Blunt ends: same length sequences or DNA
section cut in half
– Joining of two blunt ends is ligation
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
– Specific enzymes have specifc temps and times
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
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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
Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA
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
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, EZNA)
Plasmid preps
• Spectrophotometer determination of culture
density. Take OD600 of culture (equal to about
8x108 cells/ml
• 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 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
Lambda DNA
• Lambda DNA
comes from a
bacteriophage
• Genomic DNA of
Lambda is well
studied and used in
research as a size
markers for DNA
pieces
• Arrow mark HindIII
restriction sites
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
•Same thing happens in all tubes
•Read gel from top to bottom looking at all four
lanes to get the sequence.
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
Types of RNA
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Messenger RNA (mRNA)
Tranfer RNA (tRNA)
Ribosomal RNA (rRNA)
Signal Recognition Particle RNA (SRP RNA)
Small Interfering RNA (siRNA) – gene reg
Micro RNA (miRNA) – gene reg.
RNA Interference (RNAi)
• Dicer enzyme cuts dsRNA up into smaller
siRNA which then complex into the RNAinduced silencing complex (RISC) which then
cuts up the mRNA
• dsRNA can be engineered
so that genes can be
systematically shut down
Reverse Transcriptase PCR
• Use reverse transcriptase
to make a DNA copy of
mRNA
• Copy called cDNA
• Allow scientists to study the
level of gene expression in
a cell
Northern Blots
• Run mRNA on a gel
• Transfer it to nitrocellulose
membrane
• Add labeled cDNA probes to
the membrane and hybridize
the probes to the RNA
• Allows you see what genes are expressed
DNA Microarray (Chip)
• Adhere genes to chip
• Collect mRNA from cells
• Make labelled cDNA
from mRNA (red + green)
• Add cDNA to chip
• Measure signal