Intro To Genomics

Exam 2 Review

Lecture 11 — Molecular Tools: DNA cloning; PCR; Sanger Dideoxy Sequencing

Restriction Enzymes

Restriction Enzymes are also known as restriction endonucleases. They are isolated from bacteria and are used to restrict foreign DNA. What this means is that they cleave foreign DNA present in a cell in order to keep it from being expressed. All restriction enzymes cut DNA twice, once at each sugar-phosphate backbone of the DNA double helix being targeted.

Modification Enzymes

Modification enzymes, a methylase, protects host DNA from restriction enzymes/endonucleases. This keeps them from impeding the expression of legitimate DNA. Modification enzymes work by keeping restriction enzymes/endonucleases from impeding expression of legitimate host DNA through the modification of the DNA such that cleavage is blocked.

Restriction Modification System

Together restriction enzymes/endonucleases and modification enzymes/methylases form the restriction modification system. The RM system utilizes restriction enzymes/endonucleases in order to protect prokaryotic organisms from foreign DNA by cutting/cleaving it. The RM system protects host DNA from this mechanism with modification enzymes to prevent cleavage.

Types of Restriction Enzyme Modification Systems

Type I: This was the first type of restriction enzyme modification system that was discovered, these contain both the methylase(modification enzyme) and endonuclease(restriction enzyme). They work by binding to a recognition site, which can then be methylated. After this the DNA will loop back and cleaved at the cut site. The cut site for Type I restriction enzymes can be up to 1000 bases from the recognition site.

Type II: This restriction enzyme modification system is the type normally used in experiments. Unlike Type I, Type II restriction enzymes have separate molecules for the endonuclease and methylase. Also unlike Type I, Type II will always have the cut site within the recognition/binding site and the cut will always happen in the same location. Similar to Type I, Type II will have the same recognition site for both the endonuclease and the methylase. As well a nuclease will not bind to the recognition site if the site has already been methylated.

Type III: There is a third type of RM system that involves the use of “R” and “M” proteins. These proteins together form a complex of modification and cleavage.

Recognition Site Properties

Recognition sites are referred to as “n-base” cutters, where n is the length of the recognition site. Recognition sites can be 4 to 12 bases long. The average digestion frequency is a function of n. 4-base cutters have an average digestion frequency of 256 base pairs. 6-base cutters have an average digestion frequency of 4096 base pairs. And in general n-base cutters have an average digestion frequency of 4^n. Recognition sites are also normally palindromic. The reason for this is because this means that the enzyme is able to attach to and cut both DNA strands.

Restriction Enzyme Uses

Restriction enzymes have many uses, particularly in the areas of gene engineering(i.e., molecular cloning) and restriction mapping.

Molecular Cloning

Molecular cloning involves the use of restriction enzymes in order to insert DNA fragments into plasmid vectors in order to produce recombinant DNA. This recombinant DNA is then introduced into a host organism that is easy to grow, such as E. coli. As these host organisms are grown a large population of the recombinant plasmids are replicated alongside the host organisms DNA.

Steps in Molecular Cloning

1. Choice of host organism and cloning vector: Typically E.coli and a plasmid cloning vector. However E.coli and plasmids are not the only organisms and vectors available for molecular cloning
2. Preparation of vector DNA: Restriction enzyme is applied to the cloning vector to facilitate insertion of foreign DNA to be cloned. Normally to ensure compatibility the foreign DNA and cloning vector are cleaved with the same enzyme.
3. Preparation of DNA to be cloned: DNA is extracted from the organism being studied. Then the DNA is purified in order to remove proteins, RNA, and other small molecules. Purified DNA is then treated with the restriction enzyme in order to generate DNA fragments with ends that are compatible with the cloning vector. In some cases DNA linkers can be added to incompatible fragments in order to make them compatible with the cloning vector.
4. Creation of recombinant DNA: DNA produced in preparation steps for the cloning vectors and target organism are mixed together along with DNA ligase in order to link the fragments together. This linking is known as ligation. Caveat: DNA sequences that aren’t the desired amalgamation of vectors + target DNA will be present. These are removed in
5. Introduction of recombinant DNA into host organism:
6. Selection of organisms containing recombinant DNA:
7. Screening for clones with desired DNA inserts and biological properties:

Restriction Mapping

Gel Electrophoresis

Gel Electrophoresis is a technique used in order to separate and analyze macromolecules and their associated fragments based on their size and/or charge. The specific process is as follows.

1. Creation of a matrix of either agarose or polyacrylamide
2. Forcing of DNA fragments through matrix using electric current: A positive electrode is placed at the bottom of the gel matrix, which attracts negatively charged DNA. Smaller fragments will move through the matrix faster, leading to them being further into the “Gel” matrix.
3. Analysis: Visualization of distance traveled by DNA fragments.

Visualization of DNA fragments

Ethidium bromide is a molecule with properties that allows it to bind to DNA molecules. Specifically it is intercalating (def: insert between layers layers in a crystal lattice, geological formation, or other structure) allowing it to insert between the nitrogenous bases in the DNA molecule. The other property of Ethidium bromide is that it fluoresces under ultraviolent light. This allows for visualiztions of the relative number of fragments of varying sizes.

Ethidium Bromide Stain of DNA in Agarose Gel under UV light.

Membrane Hybridization Assay

This assay is used to detect DNA and RNA. It works by hybridizing a DNA or RNA probe (labeled molecule — radioactive or fluorescent tag used for detection). Then non hybridized DNA/RNA is removed leaving only hybridized that can be inspected to detect nucleic acids.

Genomic and cDNA Libraries

cDNA is DNA produced from fully transcribed mRNA. This means that it contains only the expressed genes of an organism. cDNA libraries are collections of cDNA. Alternatively genomic DNA libraries are collections of all genomic DNA for a single organism. The benefits of cDNA libraries are that they allow for easy identification of regions that are expressed genes. This is also the drawback. Varying tissues have different gene expression profiles, so cDNA libraries must be made for each tissue. As well cDNA lacks information about enhancers, introns, and various regulatory elements.. all of which is present in genomic DNA libraries.

Construction of Genomic/cDNA Libraries

Polymerase Chain Reaction(PCR); Non-Bacterial Cloning

Sanger Dideoxy DNA Sequencing

1. Procedure
2. Advantages
3. Disadvantages

DNA Labeling with Specialized Nucleotides

Automated Sequencing

Sequencing Strategies

1. Linear Sequencing
2. Shotgun Sequencing

Physical Mapping

Human Genome Sequencing Project