Non amplification techniques

8/7/2019 Non amplification techniques

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D/RNA Non-

Amplification

Techniques


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Whole genome RFLP

Southern

Northern Microarray

Non-Amplification Techniques


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RFLP Restriction Fragment Length Polymorphism

RFLP was developed at the late 70s due to the discovery of

restriction enzymes (REs; or called as restriction endonucleases)

from bacteria.

RE acts as molecular scissors to cut DNA molecules at specific

sequence.

e.g. EcoRI recognizes sequence GAATTC.

Daniel Nathans and Hamilton Smith received Nobel Prize in

Medicine (1978) for the discovery of restriction endonucleases,

leading to the development of recombinant DNA technology.


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1962 Arber and Dussoix- postulated certain bacterial strains contain restriction

enzymes able to cleave l Phage DNA, but protect their own DNA

1968 Arber and Linn

- demostrated restriction nuclease activity ofEcoB restriction

enzymes

1960s Hamilton Smith, Werner Arber

- HindII first type II restriction Enzyme identified

- discovered the recognition site for the HindII

- won the Noble Prize for Medicine and Physiology1970s Daniel Nathans

- pioneered the use of restriction enzymes


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Arber and Dussoix Certain bacterial strains were able to prevent the invasion of

foreign DNA by cleaving the foreign DNA before replicating into

the host DNA and this was done with a specialized enzyme called

endonucleases

These special enzymes contained a strain-specific modification

system.

This was the bacterias defense mechanism to prevent the

invasion of the virulent DNA into their genome, restricting phage

infection

Unfortunately, some of the phages DNA is modified, replication

occurs and the phages is able to infect a second host, losing the

susceptibility in the original host.


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Naming of REsNaming of REs

Restriction enzymes are named based on bacteria in which

they are isolated in the following manner:

e.g. EcoRI:

E Escherichia (genus)

co coli (species)

R RY13 (strain)

I First identified (order identified in bacterium) BamHI (Bacillus amyloliquefaciens); HindIII (Haemophilus

influenzae); TaqI (Thermus aquaticus)


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Commonly used RE, Recognition Sites


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Type ICleaves randomly does not cleave where bound

Type II

Cleaves at a specific site and cleaves where bound

Type III

Cleaves at a specific site but does not cleave where

bound

Types of Restriction Enzymes


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Symmetrical cuts

Blunt ends when the restriction enzyme cuts

between the same two base pairs

Sticky ends when the restriction enzyme cuts

leaving either a 5 overhang or a 3 overhang

Not symmetrical cuts

Occurs in longer strands of DNA ; BstXI

Types of recognition Sequence and Cut


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Over 3000 REs have been studied in detail, and more than

600 of these are available commercially.

44--base cuttersbase cutters 66--base cuttersbase cutters

HpaII CCGG HindIII AAGCTTRsaI GTAC EcoRI GAATTCTaqI TCGA DraI TTTAAA

AluI AGCT BamHI GGATCCHinfI GANTC BglI AGATCTDdeI CTNAG ClaI ATCGAT


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HpaII CCGG HindIII AAGCTTRsaI GTAC EcoRI GAATTCTaqI TCGA DraI TTTAAA

AluI AGCT BamHI GGATCC

HinfI GANTC BglI AGATCTDdeI CTNAG ClaI ATCGAT

Some REs produce blunt ends and others produce sticky

ends.

CfoI 5G/CGC3 5G CGC33GCG/C5 3CGC G5

EcoRV

Sticky

ends

5GAT/ATC3 5GAT ATC33CTA/TAG5 3CTA TAG5

Blunt

ends


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RFLP Technique

DNA Extraction

Digestion of DNA with Restriction enzyme

Separation of the restricted fragments with Agarose gel

electrophoresis

Southern Blotting

Hybridization

RFLP scoring with autoradiogram


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Disease Status

Forensic DNA fingerprinting

Paternity cases

Genetic Mapping

RFLP applications


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This segment of DNA was cut with Eco I; the cut produces three

restriction fragments, but only one contains the target sequence whichcan be bound by the complentary probe.

RFLP Production


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SOUTHERN BLOTTING

The technique was developed by E.M.Southern in 1975.

The Southern blot is used to detect thepresence of a particular piece of DNA in a

sample.

The DNA detected can be a single gene, orit can be part of a larger piece of DNA such

as a viral genome.


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There are 2 important features of

hybridization:

The reactions are specific-the probes will only

bind to targets with a complementary

sequence.

The probe can find one molecule of target in a

mixture of millions of related but non-

complementary molecules.


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Steps for hybridization

1. The mixture of molecules is separated.

2. The molecules are immobilized on a matrix.

3. The probe is added to the matrix to bind to the

molecules.

4. Any unbound probes are then removed.

5. The place where the probe is connected

corresponds to the location of the immobilized target

molecule.


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Preparing the samples and running the gel

Southern transfer

Probe preparation

Prehybridization

Hybridization

Post-hybridization washing

Signal detection

Flow chart of Southern hybridization


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Nucleic acids have a net negative charge and will move

from the left to the right. The larger molecules are held up

while the smaller ones move faster. This results in a

separation by size.

SOUTHERN BLOTTING


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1) Electrophoresis- takes advantage of the molecules negative charge.

2) Capillary blotting-fragments are eluted from the gel and

deposited onto the membrane by buffer that is drawn through the

gel by capillary action.


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Southern transfer


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Northern blotting or Northern hybridization

Technique for detecting specific RNAs

separated by electrophoresis by hybridization

to a labeled DNA probe.


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Prepare RNA samples and run RNA gel

Northern transfer

Probe preparation

Prehybridization

Hybridization

Post-hybridization washing

Signal detection

The flow chart of Northern hybridization

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Non amplification techniques