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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 - PowerPoint PPT Presentation
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Molecular Biology IICommon Techniques
DNA/RNA QuantitationGel Electrophoresis
Aspects to Cover
Restriction Endonuclease DigestionDNA LigationDNA/RNA PolymerasesReverse Transcription (RT)Plasmid VectorsCloningSouthern/ Northern BlottingPrimersPolymerase 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
Can create ‘sticky’ or ‘blunt’ ends
SmaI
G G G C C C
C C C G G G
C C C
C C C
G G G
G G G
Restriction EndonucleasesRestriction Enzyme
DNA Sequence Recognized
EcoRI5'G A A T T C3'C T T A A G
BamHI5'G G A T C C3'C C T A G G
HindIII5'A A G C T T3'T T C G A A
MstII5'C C T N A G G3'G G A N T C C
TaqI5'T C G A3'A G C T
NotI5'G C G G C C G C3'C G C C G G C G
AluI5'A G C T3'T C G A
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 – 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 TranscriptaseUses RNA as template for DNA synthesis
5`-CGATCGGATCCAGCTGGACGCTAGCGTAAAAAAAA-3`5`-CGAUCGGAUCCAGCUGGACGCUAGCGUAAAAAAAA-3`TTTTTT-5`3`-GCTAGCCTAGGTCGACCTGCGATCGCATT
RT
Reverse Transcriptase
RNA-dependent DNA polymerase: synthesises DNA from RNA template
Major constituent of retroviruses: facilitates insertion of viral genome into host genome
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 hexamergene-specific - designed for target gene
Reverse Transcriptase enzyme: M-MLV, AMVRNaseH
RNA must be heated to 65oC to remove secondary structure
Used predominantly to generate complementary DNA (cDNA) as first step in RT-qPCR mRNA quantitation or in cloning
C
C
C
CCG
G
G
G
G
A
A
A
AU
U
U
A
Plasmid VectorsSmall 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
When the host cell divides, copies of the vector are passed to the progeny
Plate bacterial host on agar and allow time for multiple cell divisions to form a colony (clone). Each cell in the clone contains one or more copies of the vector and gene. The initial fragment is now said to be cloned.
CloningA fragment of DNA is ligated into a circular DNA molecule (vector), creating a recombinant DNA molecule.
The vector is transformed into a host cell (bacteria)
The bacteria replicates the vector
The plasmid and the insert can be then isolated in bulk for subsequent procedures – further cloning, sequencing, Southern/Northern blotting etc
Hybridise labelled probe (from target gene) to membrane and wash away unbound probeOriginally named after Edward Southern
Visualise bound probe and determine complexity of gene by size and number of fragments
Southern BlottingIsolate genomic DNA from organism of interest
Digest DNA with restriction enzymes and electrophorese
Transfer fractionated DNA to nitrocellulose membrane by capillary transfer in alkaline buffer to denature DNA strands
Each different genomic digest has two hybridising fragments suggesting two copies of the gene (unless each restriction site occurs in the probe sequence)
Each different genomic digest has one hybridising fragment suggesting a single copy of the gene
Enables detection of specific DNA sequences amongst an unknown group of sequences – helps assess gene complexity and copy number within a genome
Enables detection of related but not identical sequences by variation in wash stringency
Northern BlottingEarly 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
If targeting a specific gene, must not be complementary to any other region in template
Can either target a specific gene (PCR) or randomly prime (RT)
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)
Taq polymerase synthesizes DNA complementary to template in 5` to 3` directionTargeted DNA replication using thermostable DNA polymerase
Each cycle of PCR doubles the number of progeny DNA duplexes (which can then act as template as well)
1 cycle = 21 copies of starting template25 cycles = 225 copies of starting template
The use of two primers allows targeting of specific sequences
Polymerase Chain Reaction
Primers are complementary to opposite strands of target region but not complementary to any other sequences
DENATURE
94oC
ANNEAL PRIMERSEXTEND STRANDS
50 – 65oC
72oC
94oC
50-65oC
72oC
PCR Requirements
dNTPs – for incorporation into elongating DNA fragment
Enzyme – Taq polymerase or equivalent
primers – forward and reverse pairgene specific0.2 M – 10 M
MgCl2 – essential for primer binding0.5 – 4 mM
Buffer – to maintain pH
Quantitative PCR (qPCR)Regular PCR involves performing the reaction and electrophoresing the final product – not reflective of starting amount
Real-time qPCR enables assessment of the reaction after each cycle
Cycle number0 35
100
ConventionalPCR
RealtimeqPCR
Pro
duct
Am
ount
Machine measures amount of PCR product after each cycle by measuring the intensity of fluorescence
Fluorescence from excitation of SYBR Green molecule by laser, SYBR Green only fluoresces when binds to dsDNA
Quantitative PCR (qPCR)
Cycle number0 35
100
Create a standard curve with 10-fold serial dilutions of PCR product – assign arbitrary values
Compare values from standards with values for unknown sample
Pro
duct
Am
ount STD 1: 1,000,000
STD 2: 100,000STD 3: 10,000STD 4: 1,000STD 5: 100STD 6: 10
Sample: 6,592
Regulation of Leptin Expression
Leptin (Ob)
Adipocytes
Hypothalamus
Ob-R NPY-ve
-ve
-ve
Regulation of Leptin Expression
Human placenta also source of leptin
BeWo cells (human choriocarcinoma) used as an in vitro model of placental function
hOb gene structure
Gong et al. (1996) JBC 271:3971
Con
Leptin mRNA in primary placental cultures
Coya et al. (2001) Biol. Reprod 65:814
FS
K
Regulation of Leptin ExpressionTreated 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
Effect of Forskolin on BeWo Expression of Leptin
0
10
20
30
40
50
Control Forskolin
Rel
ativ
e E
xpre
ssio
n of
lept
in
Forksolin treatment increases leptin mRNA expression in BeWo placental cells 20 fold