METHODS IN MOLECULAR BIOLOGY

List of covered topics:

• Recombinant DNA, cloning vectors, gene libraries.
• Purification and labeling of nucleic acids, hybridization.
• DNA modifying enzymes, cloning strategies, DNA sequencing.
• PCR applications, degenerate, nested, touch-down PCR, RACE.
• Analyses of mRNA, S1 mapping and primer extension.
• Analyses of gene expression at the mRNA level: in situ hybridization, RT-PCR,
quantitative real-time RT-PCR, microarray technology.
• Extraction and separation of proteins. Design and preparation of recombinant proteins
in E. coli, expression vectors, fusion and tagged proteins.
• Antibodies, immunodetection methods. Analyses of gene expression at the protein
level: western blotting, immunocytochemistry.
• Protein-protein interaction analyses: GST pull-down assays, immunoprecipitation,
yeast two-hybrid system.
• Protein-DNA interaction analyses: EMSA, DNase protection assays.
• Analyses of transcriptional regulation in cell transfection systems.
• Analyses of gene expression and function in vivo using transgenic methods in
eukaryotic models (Drosophila and C. elegans).
• Open topic or discussion.
Přehled transkripce a translace v eukaryotické buňce
Tvorba nukleových kyselin
Single Stranded Nicks in DNA

Hydrolysis of
this ester bond
OH
O-
O P OH
O

H2O
Restriction/Methylation Enzyme
 Eco RI Restriction Enzyme

Single stranded “nick”

• First restriction enzyme from Escherichia coli, so Eco R1

Restriction Enzyme Recognition Sites

Restriction sites are general palindromic:

“Able was I, ere, I saw Elba”

5’-GGATCC-3’
Bam H1 site:
3’-CCTAGG-5’
 Restriction Enzyme Recognition Sites

BglII 5’ A-G-A-T-C-T
T-C-T-A-G-A 5’

Sau3A 5’ G-A-T-C All these sticky ends

C-T-A-G 5’ are compatible

BamHI 5’ G-G-A-T-C-C
C-C-T-A-G-G 5’

Isoschizomers: In certain cases, two or more different enzymes may

recognize identical sites. (e.g. MboI also cleaves at GATC, and so is an
isochizomer of Sau3A.)
 Frequency of cutting of recognition enzymes

Sau 3A (GATC) cuts (¼)(¼)(¼)(¼) = once every 256 base pairs

(assuming G/C = A/T, which is often does not)

BamH1 (GGATCC) cuts (¼)(¼)(¼)(¼)(¼)(¼) = once every ~4Kb

HindII (GTPyPuAC) cuts (¼)(¼)(½)(½)(¼)(¼) = once every ~1Kb

 “Sticky” ends
5’ overhang (EcoRI)
5’-GAATTC-3’  5’-G-OH PO4-AATTC-3’
+
3’-CTTAAG-5’ 3’-CTTAA-PO4 HO-G-5’

3’ overhang (PstI)
5’-CTGCAG-3’  5’-CTGCA-OH PO4-G-3’
+
3’-GACGTC-5’ 3’-G-PO4 HO-ACGTC-5’

“Blunt” ends

5’ overhang (SmaI)
5’-CCCGGG-3’  5’-CCC-OH PO4-GGG-3’
3’-GGGCCC-5’ 3’-GGG-PO4
+ HO-CCC-5’
 Ligation of compatible sticky ends

Human DNA cleaved with EcoRI Corn DNA cleaved with EcoRI
5’-C-G-G-T-A-C-T-A-G-OH
3’-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4
+ PO4-A-A-T-T-C-A-G-C-T-A-C-G-3’
HO-G-T-C-G-A-T-G-C-5’
Complementary base pairing

5’-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3’
3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’

+ DNA Ligase, + rATP

5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’
3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’

recombinant DNA molecule

 Agarose Gel Electrophoresis

_
DNA is negatively
charged from the
phosphate backbone
Agarose mesh
+

Visualize DNA with ethidium

bromide – fluoresces orange
ONLY when bound to DNA
1 2 3 4
1
10 kb
1 2 3 4
1
10 kb

2 Eco R1
3 kb 7 kb
1 2 3 4
1
10 kb

2 Eco R1
3 kb 7 kb

3 PstI
4 kb 6 kb
PstI

6 kb 4 kb
1 2 3 4
1
10 kb

2 Eco R1
3 kb 7 kb

3 PstI
4 kb 6 kb
PstI

6 kb 4 kb

4 Eco R1 PstI
3 kb 1 kb 6 kb
 Plasmid vectors

• Circular DNA molecules that replicate independently of E.

coli chromosome.
• Are present in various copy per cell - Some are very high copy
(can be > 100 per cell); Others are low copy (1-25 per cell).
• Three key features of plasmid vectors:
1) Origin of replication (e. g. ColE1, very high copy 500
copies per cell).
2) Antibiotic resistance (or other selectable marker).
3) Multiple cloning site (often embedded in a LacZ reporter
for ease of selecting inserts)
 Useful Plasmid
Features
• Relaxed Replication
• Selectable Markers
• Streamlined
• Polylinker or MCS
• Identification of
Recombinants
• most derived from
pUC or pBR322
Multiple Cloning Site:
|SacI| |ScII| |XbaI||SpeI||BamH||SmaI||PstI||EcRI||EcRV||HIII||ClaI| |SalI||XhoI| |KpnI|
GAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACC
CTCGAGGTGGCGCCACCGCCGGCGAGATCTTGATCACCTAGGGGGCCCGACGTCCTTAAGCTATAGTTCGAATAGCTATGGCAGCTGGAGCTCCCCCCCGGGCCATGG
 pBluescript origin of
A widely used plasmid
replication
cloning vector

ampicillin
resistance
gene
MCS

MCS, Multiple Cloning Site

 Ligation Reaction
• mix foreign and vector DNA in
presence of DNA ligase
• optimal ratios of vector to insert generally
1.5-2:1
• intermolecular base-pairing can occur
between compatible overhangs
IV. Kinases and Phosphatases
• add or remove phosphate groups and the 5’ ends of
DNA or RNA.
Kinase +ATP
HO-GATC… PO4-GATC…
Phosphatase

be a
m

ha
A

ta
m

al p
ga
(P)-(P)-(P)- O

- the enzyme is not sequence-specific

Intramolecular vs. Intermolecular
Removal of 5’-PO4 Prevents
Vector Self Ligation
 CLONING
TERMINI REQUIREMENTS COMMENTS
Phosphatase treatment of Restriction sites at junctions preserved.
Identical
linear plasmid improves Both orientations of insert DNA possible.
Overhangs efficiency. Tandem copies of insert possible.
High concentrations of DNA Restriction sites at junctions often
Blunt-end and ligase needed. eliminated. Tandem copies of insert DNA
Phosphatase treatment. possible. Both orientations possible.
Restriction sites at junctions preserved.
Purification of double-cut
Different Background of non-recombinants is low.
plasmid increases
Overhangs efficiency.
One possible orientation of insert. Tandem
copies unlikely.
 Purification of Plasmids
Takes advantage of distinct topological state of plasmids.
- plasmids will be covalently closed, negatively wound circles when E. coli is
lysed.
- chormosomal DNA will be sheared into linear, non-topologically constrained
fragments (because so big).

This difference can be exploited to allow

purification of plasmids:
- difference in binding ethidium bromide,
leading to different densities (CsCl
banding,
right).
- Different rater of re-associate of two strands
following denaturation by boiling or
alkaline
treatment
Generic rDNA Protocol
• prepare foreign DNA
• prepare vector
• ligate foreign DNA and vector
introduce rDNA into host
– heat-shock
– electroporation

Transformation
• incubate ligation mixture
with ‘competent cells’
– cells pretreated to enhance
DNA uptake
• treat according to method
– 40-41o for 1-2 minutes
– brief pulse of high voltage
 Bacterial Transformation with a Plasmid
chromosome
E. Coli cell
Amps

Ampr
Permeablize membrane
with Ca2+ and heat shock

E. Coli cell
Ampr + plasmid

Select for growth in the presence of ampicillin

• select for transformants
with antibiotic
• electroporation = 109-1010
colonies/g DNA
• heat-shock = 105-109
colonies/g DNA)
 Identifying Recombinants
• based on interruption of a gene
• eg., lacZ gene = -galactosidase
• intact -galactosidase produces
blue color in presence of X-gal
-complementation or blue-
white screening