rflp wrt restriction enzymes and pcr

Restriction Fragment Length

Polymorphism

RFLP- “rif-lip”

RFLP’s contribution . . . . .• Restriction fragment length polymorphism

(RFLP) markers were regarded as the first shot in the genome revolution, marking the start of an entirely different era in the biological sciences.

• RFLP was the most popular approach for analysis of genetic variation during the entire 1980s.

• RFLP is based DNA fragment lengthdifferences after digesting genomic DNA withone or more restriction enzymes.

• DNA is digested by one or more restrictionenzymes and separated on an agarose gel.

• The DNA in the gel transferred tonitrocellulose or nylon membranes.

Intro. . .

• The specific DNA locus with a potential fragmentlength difference is characterized byhybridization to a probe, a radioactively labeledDNA or ribonucleic acid (RNA) molecule withsequence similarities to the locus of interest.

• After hybridization, the nonspecific probes mustbe washed away leaving only hybridized probesto the specific locus.

• The membrane is then exposed to a piece of X-ray film for autoradiography to visualize the DNAbands.

Technology Advances - Development of RFLP

• Two specific technological advances that set

the foundation for RFLP were —

– The discovery and application

of restriction enzymes and

– The development of DNA hybridisation.

The History of RE. . . .• The phenomenon was first identified in work done in the laboratories of Salvador

Luria and Giuseppe Bertani in early 1950s.

• It was found that, for a bacteriophage λ that can grow well in one strainof Escherichia coli

• In the 1960s, it was shown in work done in the laboratories of WernerArber and Matthew Meselson that the restriction is caused by an enzymaticcleavage of the phage DNA, and the enzyme involved was therefore termed arestriction enzyme.

• The restriction enzymes studied by Arber and Meselson were type I restrictionenzymes, which cleave DNA randomly away from the recognition site.

• In 1970, Hamilton O. Smith, Thomas Kelly and Kent Wilcox isolated andcharacterized the first type II restriction enzyme, HindII, from thebacterium Haemophilus influenzae.

• Restriction enzymes of this type are more useful for laboratory work as theycleave DNA at the site of their recognition sequence.

• For their work in the discovery and characterization of restriction enzymes, the1978 Nobel Prize for Physiology or Medicine was awarded to Werner Arber, DanielNathans, and Hamilton O. Smith.

Restriction enzymes- Restriction endonucleases

• Proteins produced by bacteria that cleave DNAat specific sites along the molecule.

• In the bacterial cell, restriction enzymescleave foreign DNA, thus eliminating infectingorganisms.

• Restriction enzymes can be isolated frombacterial cells and used in the laboratory tomanipulate fragments of DNA.

• They are indispensable tools of . . . . .

The obvious question . . ..

Why the restriction enzymes do

not digest bacterial DNA ??????

“The answer is that the bacteria also harbor aset of defense weaponry containing so-calledrestriction enzyme modification systems.Usually, organisms that make restrictionenzymes also make a companion modificationenzyme (DNA methyltransferase) that protectstheir own DNA from cleavage. These enzymesrecognize the same DNA sequence as therestriction enzyme they accompany, butinstead of cleaving the sequence, theydisguise it by methylating one of the bases ineach DNA strand.”

The vast nature of RE

• To date, more than 10,000 bacteria specieshave been screened for the existence ofrestriction enzymes; more than 2,500restriction enzymes have been found withmore than 250 distinct specificities.

• Occasionally enzymes with novel DNAsequence specificities are still found, but mostnow prove to be duplicates (isoschizomers) ofalready discovered specificities.

Isoschizomers ???

• Isoschizomers are pairs of restriction

enzymes specific to the same recognition

sequence.

• For example, SphI (CGTAC/G) and BbuI

(CGTAC/G) are isoschizomersof each other.

• Type I and III enzymes are similar in that bothrestriction and methylase activities are carried out by one large enzyme complex,

• in contrast to the Type II system, in which the restriction enzyme is independent of its methylase.

• Type II restriction enzymes also differ from the other types in that they cleave DNA at specific sites within the recognition site; the others cleave DNA randomly.

Types of RE

The palindromes. . . • Generally speaking, Type II restriction

enzymes recognition sites are palindromes.

• A palindrome read from both sides yields thesame sequence of characters (e.g., 121, IFFI,ABA).

• However, for a DNA sequence, a palindromerefers to reading the sequence from bothstrands from 5-3. For instance, the EcoR1 siteis 5-GAATTC-3; and its complementary strandshould also read 5-GAATTC-3.

The nomenclature of RE . . .

• Restriction enzymes are named by using the first letter of the genus name and the first two letters of the species name from which they were isolated.

• Often, additional letters are used to designate the strains from which they were derived, or the chronological order in which the enzyme was isolated from the species.

For example . .

• The enzyme EcoRI is produced by Escherichia coli strain RY13;

• Pst I was isolated from Providencia stuartii;

• Hind III was isolated from Haemophilusinfluenza, and

• Not I was isolated from Norcardia otitidis-caviarum

• Restriction enzymes have recognitionsequences of 4, 6, or 8 base pairs.

4-base pair

Taq 1,

, Hpa II

Msp I

6-bp cutters

EcoR1,

Hind III,

Bam H1

8-bp cutters

Not I

Sfi I.

• The odds or frequency of restriction enzymes digestingDNA depends on their recognition sequences.

“The shorter the recognition sequences, the higher thecutting frequency.”

• Restriction enzymes with 4-bp recognition sequencesdigest DNA at a frequency of one per 44 = 256 bp;restriction enzymes with 6-bp recognition sequencesdigest DNA at a frequency of one per 46 = 4,096 bp;restriction enzymes with 8-bp recognition sequencesdigest DNA at a frequency of one per 48 = 65,536 bp.

The frequency of restriction enzymes

• Three types of ends can be produced by Type II restriction enzymes including

– 3-overhang (protruding),

– 5-overhang, and

– blunt-ended molecules

• Choose RE wisely –

– some restriction enzymes do not digest DNA efficiently when the recognition sites are located close to the end of DNA.

– This is particularly important when incorporating restriction sites into PCR primers for cloning.

The development of DNA hybridisation

• These hybridisation techniques are usedextensively in the research laboratory fordetecting specific nucleotide sequences inDNA and RNA and are increasingly beingapplied in medicine for diagnosing diseases.

• All of the hybridization techniques startedwith a simple hybridization technique calledSouthern blot (Southern 1975).

Southern blot• A Southern blot is a method in molecular biology

of enhancing the result of an agarose gelelectrophoresis by marking specific DNAsequences.

• The method is named after its inventor, theBritish biologist Edwin Southern.

• This caused other blot methods to be named asplays on Southern’s name (for example, westernblot, northern blot, southwestern blot, etc.).

• All of these blotting techniques require the use ofmolecular probes.

Probe

• Agent that is used to detect the presence of amolecule in the sample.

• For Southern blot, the probe is a DNA sequencethat is used to detect the presence of acomplementary sequence by hybridization with aDNA sample.

• Probes are needed to screen for a gene ofinterest, to determine genomic structure andgene copy numbers, to analyze gene expression,or to validate allelic amplification in PCR.

Principle and Molecular Basis of RFLP

• Restriction endonucleases cut DNA wherever their recognition sequences are encountered so that changes in the DNA sequence due to indels, base substitutions, or rearrangements involving the restriction sites can result in the gain, loss, or relocation of a restriction site.

• Two approaches are widely used for RFLP analysis.

Two approaches

Use of hybridization

Use of PCR

Southern blot analysis

• Traditionally, fragments were separated using Southern blot analysis (Southern 1975), in which genomic DNA is

– digested

– subjected to electrophoresis through an agarose gel

– transferred to a membrane

– visualized by hybridization to specific probes.

PCR• In recent approaches - PCR replaces the Southern

blot analysis.• If flanking sequences are known for a locus, the

segment containing the RFLP region is amplified viaPCR.

• If the length polymorphism is caused by arelatively large (approximately 100 bps dependingon the size of the undigested PCR product) deletionor insertion, gel electrophoresis of the PCRproducts should reveal the size difference.

• However, if the length polymorphism is caused bybase substitution at a restriction site, PCR productsmust be digested with a restriction enzyme toreveal the RFLP.

• With the increasing number of ‘universal’ primersavailable in the literature a researcher can targetDNA regions that are either relatively conservedor rapidly evolving, depending on the amount ofvariation observed and the taxonomic level underexamination.

• Also, PCR products can be digested withrestriction enzymes and visualized by simplestaining with ethidium bromide due to theincreased amount of DNA produced by the PCRmethod.

• If the size shift is small, polyacrylamide gels orsequencing gels should be considered rather thanagarose gels.

Inheritance of RFLP Markers

• RFLP markers are inherited in a Mendelianfashion as codominant markers.-The strengthof RFLP markers.

• Both alleles are expressed in molecularphenotypes (here, bands on gels).

• In the case of an individual heterozygous fortwo allelic RFLP patterns on alternativechromosomes, the phenotype includes bothof the patterns .

In the example, a base substitutio within the 8 kb fragment leads to the gainin of a newrestriction site. For homozygous AA, one band of 8 kb should be generated; forhomozygous BB, two bands of 3 kb and 5 kb should be generated; for heterozygous AB,three bands of 8 kb (from allele A), 3 kb and 5 kb (both from allele B) should begenerated.

• RFLP is a non-PCR based method .

• In this Method DNA is digested with restriction Enzymes.

• RFLP is the co dominant marker.

• RFLP is 1-10 loci detected.

RFLP RAPD

1-3 loci detected. 1-10 loci detected

Can detect allelic variants. Cannot detect allelic variants

Technique comparatively more reliable.

Technique comparatively less reliable.

Large quantity of purified DNA required i.e. 2-10µg.

Quantity of DNA required for analysis is small 10-50µg.

Different species specific probes are required.

Same primers with arbitrary sequence can be used for different species.

Advantages of RFLPs

• Highly robust methodology with good transferability between laboratories.

• No sequence information required.

• highly recommended for phylogenetic analysis between related species.

• Well suited for constructing genetic linkage maps.

• Simplicity—given the availability of suitable probes, the technique can readily be applied to any plant.

Disadvantages of RFLPs

• Large amounts of DNA required.

• Automation not possible.

• Low levels of polymorphism in some species.

• Time consuming, especially with single-copy probes

• Costly.

• Moderately demanding technically.

“It can detect only insertions and deletions oflarge sizes, and the gain or loss of restrictionsites. It is unable to detect the vast majority ofpoint mutations and deletions or insertionsinvolving small-sized segments because of itslow resolution using agarose gelelectrophoresis.”

Applications of RFLP:

RFLPs can be used in paternity cases or criminal cases to determine

the source of a DNA sample. (i.e. it has forensic applications).

RFLPs can be used determine the disease status of an individual. (e.g.

it can be used in the detection of mutations particularly known

mutations).

In human population genetics, geographical isolates and comparison

of genetical makeup of related species.

RFLP in fisheries. . ..

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