Restriction Endonuclease and Restriction Mapping - notes PDF

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Genetics

Restriction Endonuclease and Restriction Mapping Bacteriophage λ - Bacteriophage Lambda λ - Isolated in 1950 from E coli - dsDNA linear genome, 48kb - Injected through tail as linear DNA - Circularises or integrates in bacterial genome - Displays restricted growth in some bacterial strains Viral DNA is linear or circular and unpacked (no histones) as well being unmethylated

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Host cell (bacterial) DNA is packed (histone like proteins) and can be variably methylated

The restricted growth of phage DNA in certain strains was found to be due to enzymes that were able to cut viral DNA but not host DNA due to these differences in DNA structure, particularly methylation status These enzymes were therefore termed restriction enzymes or restriction endonucleases These exist in multiple classes as well as artificially made versions and have been used in a number of ways to cut, manipulate and clone DNA Restriction endonucleases All endonucleases recognise a specific DNA base sequence and make a double stranded cut in target DNA. These sites are palindromic (in the main) and may or may not be at the site of the cut Recognition site typically 4-8 bases long, and determines how frequent the site is in the genome Cut can be ‘blunt’ or result in ‘sticky ends’, and can have a 5’ or a 3’ overhang Some enzymes have different recognition sequences but produce the same ends Very useful when manipulating DNA, as it allows joining of different sequences using ligase from different DNA digests

Genetics

Other restriction endonucleases recognise the methylation state of DNA Msp I and Hpa II both recognise the sequence CCGG, but will cut differentially depending on whether the first, second or neither C base is methylated Using pairs of enzymes can therefore tell you something about DNA seq

Others can cut at sites distal to the recognition sequence, which can be useful in some cloning manipulations 4 types of restriction endonuclease: Type I, II, III and IV Type I - separate recognition and cleavage sites, at least 1000bp distant - asymmetrical recognition site (non palindomic) - random cleavage site, not a fixed distance from recognition site - rare and not terribly useful for DNA manipulation Type II - classical restriction endonucleases, very useful for DNA manipulation eg Sph I - palindromic recognition site, homodimeric enzymes - cleavage and recognition site co-located Type III - recognise non-palidromic sequences, cut 20-30 bases away from site eg Fok I Type IV - recognise modified (typically methylated) DNA eg Msp I All groups require further different cofactors for optimal functionality. For DNA manipulation group is largely irrelevant, it is function that is useful Restriction Mapping Restriction mapping refers to the process of determining the location of restriction sites within an unknown piece of DNA Consider the following information: You have been asked to produce a restriction map using two restriction enzymes on an unknown piece of DNA. Upon digestion you obtain the following fragments: Hind III – 2.1 kb and 0.9 kb fragments Bgl II – 1.7 kb and 1.3 kb fragments Hind III and Bgl II double digest – 1.7 kb, 0.9 kb and 0.4 kb fragments Map the restriction sites in this unknown piece of DNA and draw these

Genetics

circular piece of DNA such as a plasmid: You have been asked to produce a restriction map using two restriction enzymes on an unknown piece of DNA. Upon digestion you obtain the following fragments: EcoRI – 3.5kb fragment SphI – 2.3 kb and 1.2 kb fragments EcoRI and SphI double digest – 2.3 kb, 0.7 kb and 0.5 kb fragments Map the restriction sites in this unknown piece of DNA and draw these From the EcoRI digest we cannot tell whether there is no sites or this is a single site in circular DNA SphI digestion produces 2 fragments – 1 site if linear or 2 sites if circular If this is linear DNA, double digest would give 2 fragments (no EcoRI sites, 1 SphI site) If this is circular DNA, double digest would give 3 fragments (1 EcoRI site, 2 SphI sites) Thus, this unknown DNA must be circular.

Genetics

Mapping gets quite complicated quite quickly if considering 3 or more enzymes on linear DNA: AacI – 2 fragments 11kb + 3kb MspI – 2 fragments 8kb + 6kb HhaI – 2 fragments 10kb + 4kb AacI plus MspI double digest – 3 fragments 6kb + 5 kb + 3kb AacI plus HhaI double digest – 3 fragments 7kb + 4kb + 3kb MspI plus HhaI double digest – 3 fragments 8kb + 4kb + 2kb AacI plus MspI plus HhaI triple digest – 4 fragments 5kb + 4kb +3kb + 2kb Process remains the same – - determine the overall size (all digests total 14kb) - calculate all possible single and double digest patterns - generate final triple digest pattern

Single Nucleotide Polymorphisms

Genetics

SNP genotyping - RFLP Now imagine your SNP is in a restriction endonuclease recognition sequence: Perform a PCR to amplify the fragment around the SNP Add restriction endonuclease and digest Run products on an agarose gel to determine fragment number and size Imagine a PCR product 163bp in length 58bp in from one end is a SNP that happens to be in an SphI recognition site If SphI doesn’t cut, we should have a 163bp product (wild type) If SphI does cut, we have 2 fragments 105 and 58bp in length (homozygous mutant) If an individual has 1 copy of the SNP and 1 wild type allele, we get all 3 fragments Perform a PCR to amplify the fragment around the SNP

Genetics Add restriction endonuclease and digest Run products on an agarose gel to determine fragment number and size RFLP – Restriction Fragment Length Polymorphism Rapid, cheap but low throughput method of SNP detection If SNP is identified in a gene, can be disease causing – RFLP is an easy diagnostic test Now superseded by faster, more accurate methods such as pyrosequencing and TaqMan Principal of all SNP detection methods however Other uses:  Genetic fingerprinting Alul digest  Paternity testing DNA cloning Why the focus on circular DNA? Bacteriophage λ genome circularises on injection into bacterial host Many phage genes not required for replication Deletion of genes allows cloning of recombinant DNA instead, making an ideal vehicle Circular DNA (plasmid) readily carried and amplified in bacteria in lab Summary Restriction enzymes extremely useful in harnessing the ability to manipulate DNA No need to memorise specific restriction site sequences Knowing how to map restriction sites would be useful RFLP and subsequent SNP detection methods now a mainstay of genetics Cloning and DNA manipulation now coming full circle through genome editing...