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Molecular Biology II
Common Techniques
Aspects to Cover
DNA/RNA Quantitation
Gel Electrophoresis
Restriction Endonuclease Digestion
DNA Ligation
DNA/RNA Polymerases
Reverse Transcription (RT)
Plasmid Vectors
Cloning
Southern/ Northern Blotting
Primers
Polymerase Chain Reaction (PCR)
Quantitative RT-qPCR
Quick application of RT-qPCR in our laboratory
Restriction Endonuclease Digestion
Cut DNA at specific 4 or 6
base pair sequences
Sequences are usually
palindromic
C C C G G G
Can create ‘sticky’ or ‘blunt’
ends
G G G C C C
SmaI
C C C
G G G
G G G
C C C
Restriction Endonucleases
Restriction
Enzyme
DNA Sequence Recognized
EcoRI
5'G A A T T C
3'C T T A A G
BamHI
5'G G A T C C
3'C C T A G G
HindIII
5'A A G C T T
3'T T C G A A
MstII
5'C C T N A G G
3'G G A N T C C
TaqI
5'T C G A
3'A G C T
NotI
5'G C G G C C G C
DNA Ligation
DNA ligase can join a break in the sugar – phosphate
backbone of DNA
Polymerases
Synthesis of nucleic acids from template in the 5’ to
3’ direction
DNA-dependent DNA polymerases (replicate DNA)
DNA pol a,b,g,d,e – replication of DNA in eukaryotes (19)
DNA pol I, II, III – replication of DNA in prokaryotes
Klenow – subunit of E. coli DNA pol I
labelling fragments for Southern/Northern blot
Taq DNA pol – from Thermus aquaticus
PCR
DNA-dependent RNA polymerases (transcribes RNA)
Reverse Transcriptase
Uses RNA as template for DNA synthesis
Reverse Transcriptase
RNA-dependent DNA polymerase: synthesises DNA from
RNA template
Major constituent of retroviruses: facilitates insertion of
viral genome into host genome
5`-CGAUCGGAUCCAGCUGGACGCUAGCGUAAAAAAAA-3`
5`-CGATCGGATCCAGCTGGACGCTAGCGTAAAAAAAA-3`
3`-GCTAGCCTAGGTCGACCTGCGATCGCATTTTTTTT-5`
RT
RNAseH activity of Reverse Transcriptase degrades RNA
strand
DNA strand can act as template for second strand synthesis
Reverse Transcription (RT)
Reaction requires a number of different components:
Appropriate buffer conditions - [salt], MgCl2, dNTPs
Primers: non-specific - oligo dT, random hexamer
G A
gene-specific
for target gene
C - designed
A
G
U
G
U
C
A
C
Reverse Transcriptase enzyme: M-MLV, AMV
RNaseH
A
RNA must be heatedCto 65oGC to remove secondary
G
C
structure
U
A
Used predominantly to generate complementary DNA (cDNA)
as first step in RT-qPCR mRNA quantitation or in cloning
Plasmid Vectors
Small circular molecules of dsDNA
Frequently used for cloning due to their
ability to carry foreign DNA into bacterial
cells and create multiple copies
Contain multiple cloning sites to assist in
insertion of foreign DNA
Contain regulatory elements for
replication and antibiotic resistance
genes for selection
Used as vectors to express cloned
genes in both bacterial and eukaryotic cells
Cloning
A
When
fragment
the host
of DNA
cell divides,
is ligatedcopies
into a of
circular
the vector
DNA are
molecule
passed
(vector),
to the progeny
creating a recombinant DNA molecule.
Platevector
bacterial
host on agar
and
allowcell
time
for multiple cell
The
is transformed
into
a host
(bacteria)
divisions to form a colony (clone). Each cell in the clone
The bacteria replicates the vector
contains one or more copies of the vector and gene. The
initial fragment is now said to be cloned.
The plasmid and the insert can be then isolated in bulk for
subsequent procedures – further cloning, sequencing,
Southern/Northern blotting etc
Southern Blotting
Originally
namedDNA
after
Edward
Southern
Hybridise
labelled
probe
(from
target
gene)
to membrane and
Isolate
genomic
from
organism
of
interest
wash away unbound probe
Digest
DNA
with restriction
enzymes
and electrophorese
Enables
detection
of specific
DNA sequences
amongst an
unknown bound
group of
sequences
– helps complexity
assess gene
complexity
Visualise
probe
and
determine
of
gene
Transfer fractionated DNA to nitrocellulose membrane
by by
and copy
numberofwithin
a genome
size
and
number
fragments
capillary transfer in alkaline buffer to denature DNA strands
Enables detection of related but not identical sequences by
variation in wash stringency
Each different genomic digest has two
hybridising fragments suggesting two
Each different
genomic
copies of the gene (unless
each restriction
digest
has one
site occurs in the probe
sequence)
hybridising fragment
suggesting a single copy
of the gene
Northern Blotting
Early example of scientific humour – virtually identical to
Southern blotting but using RNA isolated from cells instead of
DNA
Determines whether a gene is transcribed, what size the
transcript is and to what extent – level of RNA expression
Important to remember that is a snapshot of expression
levels, is a combination of synthesis and degradation of RNA
Isolate RNA and electrophorese
Transfer to membrane
Hybridise with gene-specific
labelled probe
Visualise
Primers
Used in reverse transcription, sequencing, PCR
Short pieces of DNA (18-25 bp) used to “prime” for
DNA synthesis
Provide 3` OH group for strand elongation
Must be complementary to region in template
Can either target a specific gene (PCR) or randomly
prime (RT)
If targeting a specific gene, must not be complementary to
any other region in template
Polymerase Chain Reaction (PCR)
Used to clone specific sequences of DNA for further
manipulation
Used to in conjunction with reverse transcription to quantitate
levels of a specific gene mRNA (RT-qPCR)
Polymerase Chain Reaction
oC
94
Taq
polymerase
synthesizes
DNA
complementary
template
Targeted
The
use of
DNA
tworeplication
primers
allows
using
targeting
thermostable
of specific
DNA to
sequences
polymerase
in 5` to 3` direction
oC
50-65
Primers are complementary
to opposite
strands of target
region but not complementary to any other sequences
DENATURE
ANNEAL
PRIMERS
EXTEND
STRANDS
o
72 C
72oC
Each cycle of PCR doubles the number of progeny DNA
duplexes (which can then act
as template as well)
o
94 C
1
50 – 65oC
1 cycle = 2 copies of starting template
25 cycles = 225 copies of starting template
PCR Requirements
Buffer – to maintain pH
dNTPs – for incorporation into elongating DNA fragment
MgCl2 – essential for primer binding
0.5 – 4 mM
primers – forward and reverse pair
gene specific
0.2 mM – 10 mM
Enzyme – Taq polymerase or equivalent
Quantitative PCR (qPCR)
MachinePCR
Regular
measures
involves
amount
performing
of PCRthe
product
reaction
after
and
each cycle by
electrophoresing
measuring
the intensity
the final
of product
fluorescence
– not reflective of starting
amount
Fluorescence from excitation of SYBR Green molecule by
laser,
SYBR
Green
only fluoresces
when
binds
to dsDNA
Real-time
qPCR
enables
assessment
of the
reaction
after
each cycle
Product Amount
100
Conventional
PCR
Realtime
qPCR
0
Cycle number
35
Quantitative PCR (qPCR)
Create a standard curve with 10-fold serial dilutions of PCR
product – assign arbitrary values
Compare values from standards with values for unknown
sample
Product Amount
100
STD 1: 1,000,000
STD 2: 100,000
STD 3: 10,000
STD 4: 1,000
STD 5: 100
STD 6: 10
Sample: 6,592
0
Cycle number
35
Regulation of Leptin Expression
Hypothalamus
Ob-R
-ve
NPY
Leptin (Ob)
-ve
-ve
Adipocytes
Regulation of Leptin Expression
Human placenta also source of leptin
BeWo cells (human choriocarcinoma) used as an in vitro
model of placental function
FSK
Con
Leptin mRNA in primary
placental cultures
hOb gene structure
Coya et al. (2001) Biol. Reprod 65:814
Gong et al. (1996) JBC 271:3971
Regulation of Leptin Expression
Treated BeWo cells with vehicle or forskolin for 72 hours
Isolated total RNA from treated cells
Reverse transcribed RNA to complementary cDNA
– random hexamer primers
Used Realtime RT-qPCR to determine leptin transcript levels
Relative Expression
of leptin
Effect of Forskolin on BeWo
Expression of Leptin
50
Forksolin treatment
increases leptin mRNA
expression in BeWo
placental cells 20 fold
40
30
20
10
0
Control
Forskolin