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