SELECTED TECHNIQUES AND APPLICATIONS IN MOLECULAR GENETICS

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1 SELECTED TECHNIQUES APPLICATIONS IN MOLECULAR GENETICS Restriction Enzymes The Discovery of Restriction Endonucleases p , 3, 4, 6, 7, 8 Assigned Reading in Snustad 6th ed The Discovery of Restriction Endonucleases p , 3, 4, 6, 7, 8 A Restriction/Modification (R-M) System consists of 2 coordinated enzymatic functions: 1) a DNA endonuclease and 2) a DNA methyltransferase. For typical Type II systems these functions are incorporated in two separate enzymes. The two enzyme activities of any given system are coordinated because: 1) They recognize and act on the same DNA target sequence. 2) DNA methylation inhibits strand cutting by the endonuclease. Expression of endonuclease function without methylase would be suicidal for the bacterial strain. Note that methylation of only one of the 2 DNA strands (hemi-methylation) is generally sufficient to prevent genome fragmentation; otherwise, the genome would be subject to degradation immediately after replication. If only one strand is cut, the nick in the unmethylated strand will be repaired by DNA ligase. The methylases modify specific DNA bases in the restriction sites withjout altering their base-pairing properties; i.e. methylation by these enzymes does lead to base misincorporation and mutation. R-M systems are widespread, and of common occurrence among Bacteria and Archaea. I am not aware that anyone has reported a R-M system in the Eukarya. R-M systems presumably have evolved to provide bacteria with a defense mechanism against infection by bacterial viruses. Their presence may also reduce the incidence of plasmid DNA transfer by conjugation, and of genetic recombination between strains due to DNA transfer by transduction. R-M systems are named for the bacterial species and strain from which they were first isolated. Most Type II R-M systems use a DNA recognition sequence that is 4-6 contiguous bp and which displays a two-fold axis of rotational symmetry (an "inverted repeat ).

2 Note that the literary example of a palindrome in the text has bilateral symmetry. Thus, it may be misleading. NOT THIS (BILATERAL SYMMETRY): 5' A T G C C G T A 3' 3' T A C G G C A T 5' THIS: INVERTED REPEAT SEQUENCE (2-FOLD ROTATIONAL SYMMETRY): 5' A T G C G C A T 3' 3' T A C G C G T A 5' Type II endonucleases are homodimers. They not only bind a specific DNA sequence, they cleave at a precise location in that sequence. Therefore, any target DNA will be cleaved into a specific and predictable set of fragments by a given enzyme. Often, the endonuclease cuts the two DNA strands at different (but equivalent) positions within the recognition sequence, so that the restriction fragments produced from different target DNA's have identical complementary ss extensions ("sticky ends"). These properties of the Type II endonucleases that is most important for their use in creating in vitro recombinants. Development of Type II endonucleases for in vivo manipulation of DNA, particularly for molecular cloning, has been instrumental in the advent of biotechnology as we now know it. Commercial production of TypeII endonucleases is a multi-million (multi-billion?) dollar enterprise. PRACTICE EXAM PROBLEM: Computing the Expected Frequency of a Restriction Site Based on: Sequence of the restriction site %GC in target DNA Assumption that the target DNA is a random sequence Assumption that the target DNA is not methylated Example: 5 G A N Py Pu N T C 3 N = any nucleotide; Py = either Pyrimidine; Pu = either Purine If target genome is 50% GC: Freq. of site: (.25) (.25) (1) (.5) (.5) (1) (.25) (.25) = Average fragment size: 1,020 bp if target genome is 70% GC: Freq. of site: (.35) (.15) (1) (.5) (.5) (1) (.15) (.35) = Average fragment size: 1,449 bp Note that the frequency of the restriction site is highest when GC% of target DNA = GC% of restriction site.

3 Restriction Maps Physical Maps of DNA Molecules Based on Restriction Enzyme Cleavage Sites p , 26 Milestone in Genetics: Restriction Endonucleases p. 442 Assigned Reading in Snustad 6th ed Physical Maps of DNA Molecules Based on Restriction Enzyme Cleavage Sites p , 26 "Genetic Maps" vs "Physical Maps" A restriction map is a graphic representation of a DNA molecule (linear or covalently closed) that shows the identity and position of restriction sites for one or more restriction enzymes. Restriction maps allow prediction of number and sizes of restriction fragments obtained by digesting a given molecule with a given enzyme or enzymes. Restriction maps can be derived directly from the DNA sequence, or deduced from the results of restriction digests. The text, and Fig /14.15 walk you through a process for creating a restriction map that involves performing restriction digests and then analyzing the resulting restriction fragments by agarose gel electrophoresis. On the exam, you will not be required to do this. You will be required to do the reverse (which is far more straightforward). i.e. If we provide a restriction map diagram of a DNA molecule, you must be able to predict what fragments will be produced by a restriction digest involving one or several enzymes whose restriction sites are given on the map. This may involve either linear or circular DNA molecules. (The restriction map of a circular DNA molecule will be a circle rather than a straight line.) For the text problems suggested above then, you should work them backwards. Start with the solution (the restriction map) and work back to the data they give you to start with. For the simple example in the text figure, the restriction maps is: HindIII EcoRI 1.0 kb 3.0 kb 2.0 kb Note that the orientation of the map is not relevant. i.e. The map diagram above is entirely equivalent to its reverse: 2.0 kb EcoRI 3.0 kb HindIII 1.0 kb

4 Be sure that you can use a map like this to predict the results of the digests as shown in the book.

5 Agarose Gel Electrophoresis and Southern Blotting Assigned Reading Snustad 5th ed Analysis of DNA's by Southern Blot Hybridizations Assigned Reading Snustad 6th ed Analysis of DNA's by Southern Blot Hybridizations p p Southern Analysis Genomic DNA Isolation Restriction Digest Agarose gel Electrophoresis DNA Denaturation and Transfer to Nylon Membrane Sequence Detected by Hybridization to Labeled Probe Sources of Probe Synthetic Oligonucleotides cdna Cloned DNA Restriction Fragment PCR Product

6 Individual Genome Analysis and Molecular Diagnosis of Genetic Diseases Restriction Fragment Length Polymorphism (RFLP) and Microsatellite Maps p , MOLECULAR DIAGNOSIS OF HUMAN DISEASES p DNA FINGERPRINTS p , 10, Restriction Fragment Length Polymorphism (RFLP) and Short Tandem Repeat (STR) Maps p , MOLECULAR DIAGNOSIS OF HUMAN DISEASES p DNA Profiling p , 10, 12 Classes of RFLPs: Variable (+/-) Restriction Site (see Fig. 16.3/15.3) This class of RFLP arises from base pair substitutions. Variable Number Tandem Repeats (VNTR) (= "minisatellites") (See Fig /16.10) The length of the repeated sequence is base pairs. VNTR polymorphisms (alleles) arise by a mechanism involving genetic recombination (unequal crossing over), not by mutation, which is much less frequent. That is why VNTR's are so variable that they can be used to distinguish two specific individuals. Short Tandem Reapeats (STR) (="Microsatellites")

7 The length of the repeated sequence is 2-4 base pairs. Microsattelite sequences are also subject to unequal crossing over. Insertion of a transposable element between two restriction sites may also be detected as a RFLP. RFLP s are inherited according to Mendelian Principles