Practical molecular biology 8.10-.12.2012

PD Dr. Alexei Gratchev

Prof Dr. Julia Kzhyshkowska

Prof. Dr. Wolfgang Kaminski

Assistants

Tina Fuchs Martin Hahn Amanda Mickley Illya Ovsiy

Course structure

8.10 Plasmids, restriction enzymes, analytics 9.10 Genomic DNA, RNA 10.10 PCR, real-time (quantitative) PCR 11.10 Protein analysis IHC 12.10 Flow cytometry (FACS)

GroupsStudent Tutor

Alsara, Mohmmad

Tina Fuchs

Ghosh, Sambuddha

Liu, Xiaolei

Netsch, Philipp

Vasilakis, Thomas

Al Said, Samer

Martin Hahn

Hong, Jian

Manner, Andreas

Shan, Shenliang

Wan, Shan

Gu, Song

Amanda Mickley

Lasierra Losada, Maria

Mohammad, Yousuf

Sachindra

Zhang, Juanjuan

Gudima, Alexandru

Illya Ovsiy

Lee, Kuo-Ying

Mock, Andreas

Schlickenrieder, Bastian

Zhong, Weiwei

Literature

Current protocols in molecular biology Molecular Cloning: A Laboratory Manual,

Third Edition by Sambrook www.methods.info

Plasmids, restriction enzymes, analytics

Plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA.

Vector – a carrier (plasmid or other type) used for bringing target DNA fragment into a host cell.

Vector types

Vector Target fragment length

Plasmid 0-10 kb (total size up to 15 kb)

Cosmid 10-40 kb

P1 artificial chromosome (PAC) 130-150 kb

Bacterial artificial chromosome (BAC) About 300 kb

Yeast artificial chromosome (YAC) 200 kb to 2 Mb

Plasmids are essential instruments of molecular biology

Cloning and sequencing of DNA and cDNA fragments Generation of genomic and cDNA libraries Expression of recombinant proteins Generation of mutant proteins Analysis of regulatory sequences Gene targeting

Essential vector elements

Origin of replication Antibiotic resistance gene (Amp, Kan, Tet, Chl) (Multiple cloning site)

Map of pOTB7 vector showing Chloramphenicol resistance gene (CMR), replication origin (ORI) and multiple cloning site (MCS)

Optional plasmids elements

Multiple cloning site Promoter for cloned sequence Reporter gene Tag Regulatory sequences

pc DNA 3.1 ( +) EGF P6131 bp

Amp r

Neo

BHG polyA

SV40 poly A

P CM V

SV40 prom

T7

Apa I (1705)

Bam H I (930)

Bg l II (13)

Bst X I (1678)

Eco R I (1656)

Eco R V (1668 )

Ehe I (2969 )

H in dIII (912)

Kpn I (922)

Mlu I (229)

Nde I (485)

Not I (1683)

Sca I (5689 )

Sma I (2781)

Spe I (250)

X ba I (1695)

X ho I (1689 )

Important plasmid information

Replication origin defines the host bacteria: ColE1 replication origin is required for E.coli

Replication origin may define the number of plasmid copies per bacterial cell

Bacteria may lose recombinant plasmid during cultivation due to the absence of partitioning system (par). Naturally occurring plasmids contain par that ensures that every bacterial cell contains the plasmid.

Selection of the plasmid vectorCopy number

Replication origin Intended use

Replication origin of pBR322 vector restricts number of plasmid copies per cell to 30-40.

Expression of proteins in bacteria. Very useful for toxic protein or when tight control of protein amount per bacterial cell is needed.

Replication origin of pUC vector is a mutated version of pBR322 lacking Rop/Rom gene and allows up to 500 copies of plasmid per cell.

Amplification of high amounts of plasmid DNA in bacteria.Expression of high amounts of proteins in bacteria.

Selection of the plasmid vectorPurpose of use

Purpose Special vector feature(s) Example

Recombinant protein expression in bacteria

Regulated bacterial promoterTag for protein purification

pGEX4T

Recombinant protein expression in eukaryotic cells

Eukaryotic promoterTag for protein purification or detectionEukaryotic selection marker

pcDNA3.1

Analysis of eukaroytic promoter Reporter gene pGL3basic

General cloning - pBluescript KS

Restriction enzymes (endonucleases)

Cut specific DNA sequence Protect bacteria from phage infection by digesting

phage DNA after injection Cellular DNA is protected by methylation that blocks

restriction enzyme activity Restriction enzyme (RE) means restricts virus

replication Endonucleases are enzymes that produce internal cut

called as cleavage in DNA molecule

Restriction enzymes (endonucleases)

Presence of RE was postulated in 1960 by W.Arber The first true RE was isolated in 1970 by Smith,

Nathans and Arber. In 1978 they were awarded the Nobel Prize for Phylsiology and Medicine.

RE remain indispensible from molecular cloning and sequencing.

Type I enzymes cut at a site that differs, and is located at least at at least 1000 bp away, from their recognition site.

Type II enzymes recognize sites of 4-8 nucleotides and cleave DNA at the same site

Type III enzymes recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20-30 base pairs after the recognition site.

Restriction enzymes (endonucleases)

Type I enzymes cut at a site that differs, and is located at least at at least 1000 bp away, from their recognition site.

Type II enzymes recognize sites of 4-8 nucleotides and cleave DNA at the same site

Type III enzymes recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20-30 base pairs after the recognition site.

Restriction enzymes (endonucleases)

Restriction enzymes (endonucleases)

Creating genomic and cDNA libraries Cloning DNA molecules Studying nucleotide sequence Generating mutated proteins

Plasmids, restriction enzymes, analytics

Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric current applied to a gel matrix.

Ethidium bromide stained agarose gel of total RNA (1-3) and DNA ladder (M)

Plasmid preparation stage 1

1. Plasmid-containing bacteria are cultivated in liquid media, supplemented with the antibiotics for 18 h at 37°C with intensive shaking

2. Cells are harvested by centrifugation

Preparation of the lysate

3 solutions strategy1. Resuspend in hypotonic buffer with RNase (buffer P1)

2. Lyse bacteria using NaOH/SDS solution (buffer P2)

3. Neutralize NaOH and precipitate proteins using NaAc buffer (buffer P3)

Plasmid can be isolated from obtained lysate using various strategies.

Possible methods for isolation

1. Ethanol or Isopropanol precipitation

2. Silica matrix bind-wash-elute procedure

3. Density gradient centrifugation

Precipitation “quick and dirty”

1. Ethanol is added to the lysate

2. Obtained sample incubated for 30 min

3. DNA is collected by centrifugation

• Cheap• Fast

Advantages Disadvantages• Small amounts of DNA• Poor purity, not sufficient for

applications like transfection and in vitro translation

• Concentration of the plasmid can not be determined photometrically

Also known as mini prep

Silica matrix columns

1. Apply lysate on the column

2. Wash the column

3. Elute the plasmid

4. Precipitate

• High purity of the plasmid• Fast

Advantages Disadvantages• Expensive

Gradient centrifugation

1. Mix lysate with CsCl solution

2. Add EtBr

3. Centrifuge in the ultracentrifuge for 12-36h

4. Collect the plasmid

5. Precipitate

• The very best plasmid purity

• Relatively cheap

Advantages Disadvantages• Slow• Expensive equipment is needed• High concentrations of EtBr

Concentration measurement

Photometric measurement of DNA concentration

UV 260 nm

Conc=50xOD260

Important! Photometric measurement of DNA concentration can not be applied for “quick and dirty” plasmids, because of the presence of RNA rests.

Gel electrophoresis of plasmid DNA

Selection of agarose concentration

Plasmid on an agarose gel

Questions?