Plasmid DNA Isolation andRestriction Mapping

生化科 林光輝

• Plasmid DNA isolation

• Agarose gel electrophoresis

• Determine DNA fragment

size; Restriction mapping

DNA

• Contains all genetic information for that organism• Size is usually expressed in base pairs (bp)• 2 kinds of DNA:

– Chromosomal DNA• Makes up majority of DNA

• Packed tightly into chromosomes

– Plasmid DNA• Extrachromosomal DNA

• Small circular molecules, usually 2-10 kb

• Widely used in recombinant DNA technology

Isolation of DNA

• A good prep should:– Not contain cellular proteins– Not contain RNA– Be of high molecular weight

The alkaline cell lysis method

• Harvest bacterial cells

• Resuspend in Tris-EDTA (RNase, lysozyme)

• Add lysis buffer (NaOH+SDS)

• Add sodium acetate PH6.5

• Centrifuge and discard the pellet

• Further purification (special matrix)

Quality and Quantity of DNA

• Spectrophotometer1. Quantity:

A260 = DNA 1 O.D. = 50 g/ml DNA

___ O.D. x 50 g/ml x 100 (dilution) = ___ g/ml 1 O.D.

2. Quality:A280 = protein Purity = A260/A280

If ratio ≈ 2, then assume it is a good prep.If ratio > 2, then there is RNA contamination.If ratio < 1.6, then there is protein contamination.

Quality and Quantity of DNA

• Gel electrophoresis– Quantity:

Band intensity is semi-quantitative– Quality:

Look for high MW DNA single clear band• Sheared DNA indicates poor quality smear

Basic Steps in Gene Cloning

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Vector Fragmented genomic DNA or cDNAs from

chosen resource

Recombinant DNA molecules

Host cell

Transformed host cell

Amplification of recombinant molecule

Host cell division

Numerous cell divisions CLONE

Colonies of transformed host cell clones growing on solid medium

But how many individual clones needed to represent the entire genomic DNA or expressed genes of the resource?

1 ‘gene’ purifiedin a clone

Techniques of specific cleavage of DNA

• Aim: to isolate and manipulate individual genes.

• Means: Restriction endonucleases

What are Restriction endonucleases?

• enzymes that attack and digest internal regions of the DNA of an invading bacteriophage but not that of the host.

• First enzyme extracted from E. coli (cut randomly and not always close to the desired site).

Types of Restriction endonucleases

• Type I and III: Possess both cutting (restriction) and protecting activity.

• Protecting activity (modification by methylation).

• Type I cuts at random sites.

Types of Restriction endonucleases

• Type III cuts at specific sites quite near the recognition sequence.

• but may be difficult to predict.

• ATP required for source of energy.

Types of Restriction endonucleases

Type II restriction enzymes are:

• Invariably used in DNA science

• They have several advantages: – 1) has only restriction activity; modification

activity carried by a separate enzyme.

Types of Restriction endonucleases

– 2) Each cuts in a predictable and consistent manner at a site within or adjacent to the recognition sequence.

– 3) ATP not needed. Only a cofactor Mg++ is needed.

Types of Restriction endonucleases

• Type II (continued).

• Today, more than• 1200 type II have been isolated from a variety of

prokaryotic organisms

• More than 100 types are commercially available

Property of restriction enzymes

• They break the phosphodiester bonds that link adjacent nucleotides in DNA molecules.

Nomenclature of Restriction endonucleases

• 1) First letter: initial letter of the genus

name of the organism from which the

enzyme is isolated.

• 2) Second and third letter: usually initial

letters of the organism’s species name.

Nomenclature of Restriction endonucleases

• 3) Fourth letter (if any): indicates a particular strain of organism

• 4) Roman numerals: originally indicate the order in which enzymes from the same organism and strain are eluted from chromatography column.

Nomenclature of Restriction endonucleases

More often, though, indicate the order of discovery.

Examples of Type II restriction enzymes

EcoRI E = genus Escherichia

co = species coli

R = strain RY13

I= first endonuclease isolated

Examples of Type II restriction enzymes

BamHI B = genus Bacilus

am = species

amyloliquefaciens

H = strain H

I = first endonuclease

isolated

Examples of Type II restriction enzymes

HindIII H = genus Haemophilus

in = species influenzae

d = strain Rd

III = third endonuclease isolated

Type II Restriction Enzymes

Recognize symmetric DNA sequences = inverted repeats

Only need Magnesium

Cleave within recognition site

Most are 4-8 base pair long

5’-GAATTC-3’3’-CTTAAG-5’

Type II RE practical details

3 types of cuts - 5’ overhang, 3’ overhang, blunt

4, 5 or 6 bases

5’-GAATTC-3’3’-CTTAAG-5’

5’-G-3’OH 5’P-AATTC-3’3’-CTTAA-P5’ HO3’-G-5’

Type II RE practical details

3 types of cuts - 5’ overhang, 3’ overhang, blunt

4, 5 or 6 bases

5’-GAATTC-3’3’-CTTAAG-5’

5’-GAATT-3’OH 5’P-C-3’3’-C-P5’ HO3’-TTAAG-5’

Type II RE practical details

3 types of cuts - 5’ overhang, 3’ overhang, blunt

4, 5 or 6 bases

5’-GAATTC-3’3’-CTTAAG-5’

5’-GAA-3’OH 5’P-TTC-3’3’-CTT-P5’ HO3’-AAG-5’

Agarose Gel Electrophoresis

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Sample Well 25 ng 1 kb ladder0.8% Agarose

1

2

4

681012kb

Agarose Gel ElectrophoresisSybergold Detection Limit: ~0.2-0.5 ng DNA

EtBr Detection Limit: ~5-10 ng DNA

Separation Range Vs. % Agarose

Log (MW) is linearly related to mobility

100 bp

Low % has noResolving powerat low MW

1 kb

10 kb

High % has noresolving powerat high MW

Star Activity (Relaxation of Specificity)

. Under certain conditions, the enzymes are able to recognize and cleave nucleotide sequences which differ in some positions from the canonical site.

For example, under extreme reaction conditions (i.e. low ionic strength), BamHI (recognition sequence GGATCC)  is able to cleave the following sequences: NGATCC, GPuATCC and GGNTCC. This phenomenon has been called relaxed or star activity.

Star Activity

In most practical applications of restriction endonucleases, star activity is not desirable. The analysis of a great number of reports on star activity suggest the following reasons for this phenomenon:

• prolonged incubation time or a large excess of enzyme with respect to DNA;

• high glycerol concentration (>5%) in the reaction mixture or the presence of other organic solvents, such as ethanol or dimethyl sulfoxide

• low ionic strength or high pH values in the reaction buffer; • substitution of cofactor Mg2+ with other divalent cation (such as Mn2

+ or Co2+).

Isoschizomers

• Restriction endonucleases that recognize the same sequence are isoschizomers.

Avi II and Fsp I

TGC/GCA