Methods in Molecular Biology List of Covered

8/14/2019 Methods in Molecular Biology List of Covered

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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 proteinsinE. coli, expression vectors, fusion and tagged proteins.

Antibodies, immunodetection methods. Analyses of gene expression at the proteinlevel: 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.


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Pehled transkripce a translace v eukaryotick buce


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Tvorba nukleovch kyselin


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Single Stranded Nicks in DNA

Hydrolysis of

this ester bond

H2O

P

O

O OH

O-

OH


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Restriction/Methylation Enzyme


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Eco RI Restriction Enzyme

First restriction enzyme fromEscherichia coli, so Eco R1

Single stranded nick


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Restriction Enzyme Recognition Sites

Restriction sites are general palindromic:

Able was I, ere, I saw Elba

Bam H1 site:5-GGATCC-3

3-CCTAGG-5


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Frequency of cutting of recognition enzymes

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

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

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

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


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5 overhang (EcoRI)

5-GAATTC-3 5-G-OH PO4-AATTC-3

3-CTTAAG-5 3-CTTAA-PO4

HO-G-5

Sticky ends

5 overhang (SmaI)

5-CCCGGG-3 5-CCC-OH PO4-GGG-3

3-GGGCCC-5 3-GGG-PO4

HO-CCC-5

Blunt ends

3 overhang (PstI)

5-CTGCAG-3 5-CTGCA-OH PO4-G-3

3-GACGTC-5 3-G-PO4 HO-ACGTC-5

+

+

+


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Human DNA cleaved with EcoRI Corn DNA cleaved withEcoRI

5-C-G-G-T-A-C-T-A-G-OH3-G-C-C-A-T-G-A-T-C-T-T-A-A-PO

4

PO4-A-A-T-T-C-A-G-C-T-A-C-G-3

HO-G-T-C-G-A-T-G-C-5

Ligation of compatible sticky ends

+

5-A-C-G-G-T-A-C-T-A-GA-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-AG-T-C-G-A-T-G-C-5

Complementary base pairing

+ DNA Ligase, + rATP

recombinant DNA molecule

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


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Agarose Gel Electrophoresis

_

+

DNA is negatively

charged from the

phosphate backbone

Visualize DNA with ethidium

bromide fluoresces orangeONLY when bound to DNA

Agarose mesh


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1 2 3 41

10 kb


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10 kb

1 2 3 41

Eco R1

3 kb 7 kb

2


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Eco R1

3 kb 7 kb

10 kb

1 2 3 41

2

PstI

4 kb 6 kb

4 kb6 kb

PstI

3


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Eco R1

3 kb 7 kb

10 kb

1 2 3 41

2

PstI

4 kb 6 kb

4 kb6 kb

PstI

3

6 kb

PstIEco R1

3 kb 1 kb

4


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Plasmid vectors

Circular DNA molecules that replicate independently ofE.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 reporterfor ease of selecting inserts)


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Useful Plasmid

FeaturesRelaxed ReplicationSelectable Markers

Streamlined

Polylinker or MCS Identification of

Recombinants

most derived from

pUC or pBR322

|SacI| |ScII| |XbaI||SpeI||BamH||SmaI||PstI||EcRI||EcRV||HIII||ClaI| |SalI||XhoI| |KpnI|

GAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACC

CTCGAGGTGGCGCCACCGCCGGCGAGATCTTGATCACCTAGGGGGCCCGACGTCCTTAAGCTATAGTTCGAATAGCTATGGCAGCTGGAGCTCCCCCCCGGGCCATGG

Multiple Cloning Site:


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pBluescriptpBluescript

MCS

MCS,Multiple Cloning Site

ampicillin

resistancegene

A widely used plasmid

cloning vector

origin ofreplication


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mix foreign and vector DNA inpresence of DNA ligase optimal ratios of vector to insert generally

1.5-2:1

intermolecular base-pairing can occurbetween compatible overhangs

Ligation Reaction


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IV. Kinases and Phosphatases

add or remove phosphate groups and the 5 ends of

DNA or RNA.

HO-GATC PO4-GATC

Kinase +ATP

Phosphatase

O

A(P)-(P)-(P)-

- the enzyme is not sequence-specific


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Intramolecular vs. Intermolecular


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Removal of 5-PO4 Prevents

Vector Self Ligation


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TERMI ILO I G

REQUIRE M E NTS COM M E N T S

Identic lO er ngs

s t se trea tmen t f

linear lasmid im r esefficienc .

Restricti n sites at j ncti ns reser ed .

B t rien tati ns f insert DNA ssi le.Tandem c ies f insert ssi le.

Bl nt-endH igh c ncen trati ns f DNA

and ligaseneeded.hos ha tase trea tmen t.

Restriction sites at j nctions often

elimina ted . Tandem cop ies of insert DNApossi le. Bothorien tations possi le.

Different

O erhangs

rification ofdouble-cut

plasmid increases

efficienc .

Restriction sites at junctions preser ed .

Backgroundofnon-recombinants is low .

One possibleorien tation of insert. Tandemcop ies un likel .


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Purification of Plasmids

Takes advantage of distinct topological state of plasmids.

- plasmids will be covalently closed, negatively wound circles whenE. 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 strandsfollowing denaturation by boiling or alkaline

treatment


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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 enhanceDNA uptake

treat according to method 40-41o for 1-2 minutes

brief pulse of high voltage


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Bacterial Transformation with a Plasmid

E. ColicellAmps

chromosome

Permeablize membranewith Ca2+ and heat shock

Ampr

E. ColicellAmpr+ plasmid

Select for growth in the presence of ampicillin


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Identifying Recombinants

based on interruption of a gene eg., lacZ gene = F-galactosidase

intact F-galactosidase produces

blue color in presence of -gal

E-complementation or blue-white screening

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Methods in Molecular Biology List of Covered