Molbio: Analysis and Characterization of Nucleic acids and protein

Eco RI, Eco RV, Hind III

restriction enzymes

E. coli, strain R, 1st enzyme

Eco R I is isolated from

E. coli, strain R, 5th enzyme

Eco R V is isolated from

H. influenzae, strain d, 3rd enzyme

Hind III is isolated from

Gv AATTC

Eco RI recognition seq

Gv ATATC

Eco RV recognition seq

Av AGCTT

Hind III recognition seq

Restriction endonucleases

useful because they recognize and cut specific sequences in D N A

type 1

Methylation/cleavage (3 subunits) >1000 base pairs from binding site

Eco A I G A G N N N N N N N G T C A

example of type 1 restriction enzymes

type 2

Cleavage at specific recognition sites

type 3

Methylation/cleavage (2 subunits) 24 to 26 base pairs from binding site

Hin fIII CGAAT

example of type 3

palindromic

Type II enzyme recognition sites are

palindromic

they read the same 5’ to 3’ on both strands of the double helix.

sticky ends

must match (be complementary) for optimal re-ligation

blunt ends

can be re-ligated with less efficiency than sticky ends

sticky ends

can be converted to blunt ends with nuclease or polymerase

blunt ends

can be converted to sticky ends by ligating to synthetic adaptors

number of restriction sites

in restriction enzyme mapping, the number of bands indicates the

distance between restriction sites

in restriction enzyme mapping, the size of the bands indicates the

Clustered regularly interspaced short palindromic repeats (CRISPR)

• is a system that uses invading D N A as a guide to cutting.

• it is not used mostly in research applications, where synthetic RNA are designed to guide enzyme cutting (or other activities) to selected sites.

edwin southern

southern blot is developed by

southern blot

allows analysis of any specific gene or region without having to clone it from a complex background

agarose gel electrophoresis

In southern blot, The cut D N A is separated by

electrostatic and hydrophobic, electrostatic

DNA BINDING MEDIA

nitrocellulose, nylon, reinforced nitrocellulose

electrostatic and hydrophobic

nylon, nytran, positively charged nylon

Electrostatic

capillary transfer, electrophoretic transfer, and vacuum transfer

Transfer of cut D N A from the gel to the membrane can be performed in three ways:

capillary transfer

Southern first used ____, as shown, to move the D N A by capillary action from the gel to bind to the membrane.

what region is seen

Once the D N A is transferred to the membrane, the probe determines

melting temperature

The temperature at which 50% of a nucleic acid is hybridized to its complementary strand

length of DNA, GC content (%GC), Salt concentration, Formamide Concentration

Tm in solution is a function of

Tm = 4° (G C) + 2J° (A T)

Tm For short (14 to 20 base pairs) oligomers:

GC content

is the dominant factor in melting temperature for short sequences (such as PCR primers).

stringency

describes the conditions under which hybridization takes place

formamide concentration, low salt, heat

increases stringency

too low

If stringency is ____, the probe will bind at sequences other than the intended target.

nonradioactive probes

can produce color or light signals.

radioactive or chemiluminescent detection

requires exposure of the blot to autoradiography film

color

will develop directly on the membrane

autoradiography film

To see the chemiluminescent signal, the membrane is exposed to an

Northern Blot

• No restriction digestion required

• R N A is blotted to membranes

• Probes are labeled antisense R N A or D N A

• Internal/loading controls are required

RNA

primary target separated on the gel of northern blot

1:1 with 0.04 M Tris-H C l, p H 6.8, 0.1% S D S

western blot samples are treated with denaturant, such as mixing

proteins

Western blot uses ___ rather than nucleic acids.

capillary or electrophoretic transfer

in western blot, Proteins are blotted to membranes by

DNA

target of southern blot

RNA

target of northern blot

Protein

target of western blot

Nucleic acid

probe of Southern and Northern blot

protein

probe of western blot

dot blots, reverse dot blots, slot blots

blotting formats

Amplification analysis, Expression analysis (RNA), Mutation analysis

dot blots (3)

Amplification analysis, Expression analysis

slot blots (2)

amplification

test color dominates

deletion

reference color dominates

restriction enzymes

cut D N A at specific recognition sequences

restriction enzyme mapping

D N A can be characterized by

southern blot

Specific D N A regions in a complex mixture are characterized using

western blot

Specific proteins in a complex mixture are characterized using

comparative genomic hybridization

Regions of genomic amplification or deletion are characterized using