DNA cloning - Wiley-Blackwell

1 Chapter 14 DNA cloningCloning is the process of moving a gene from the chromosome it occurs in naturallyto an autonomously replicating vector. In the cloning process, the DNA is removedfrom cells, manipulations of the DNA are carried out in a test-tube, and the DNA issubsequently put back into cells. Because E. coliis so well characterized, it is usuallythe cell of choice for manipulating DNA molecules. Once the appropriate combina-tion of vector and cloned DNA or construct has been made in E. coli, the construct canbe put into other cell types. This chapter is concerned with the details of the individ-ual steps in the cloning process:1 How is the DNA removed from the cells?

2 2 How is the DNA cut into pieces?3 How are the pieces of DNA put back together?4 How do we monitor each of these steps?Isolating DNA from cellsPlasmid DNA isolationThe first step in cloning is to isolate a large amount of the vector and chromosomalDNAs. Isolation of plasmid DNA will be examined first. In the general scheme, cellscontaining the plasmid are grown to a high cell density, gently lysed, and the plasmidDNA is isolated and concentrated. When the cells are growing, the antibiotic corre-sponding to the antibiotic resistance determinant on the plasmid is included in thegrowth media.

3 This ensures that the majority of cells contain plasmid DNA. Withoutthe antibiotic selection, an unstable plasmid ( one without a parfunction) can belost from the cell population in a few can be lysed by several different methods depending on the size of the plasmidmolecule, the specific strain of E. colithe plasmid will be isolated from, and how theplasmid DNA will be purified. Most procedures use EDTA to chelate the Mg++associat-ed the outer membrane and destabilize the outer membrane. Lysozyme is added to digest the peptidoglycan and detergents are frequently used to solubilize the membranes.

4 RNases are added to degrade the large amount of RNA found in activelygrowing E. colicells. The RNase gains access to the RNA after the EDTA and lysozymetreatments. This mixture is centrifuged to pellet intact cells and large pieces of cell FYI gradientsIn traditional preparations ofhighly purified plasmid DNA,the lysate containing theplasmid DNA was mixed withthe dye, ethidium bromide,that slips between the bases ofthe double helix (intercalation)and unwinds the DNA. Theamount of ethidium bromidethat can intercalate dependson the topology of the DNAmolecule. Covalently closedsupercoiled molecules bindless ethidium bromide thanlinear DNA because the DNA isconstrained.

5 Linear molecules,such as broken chromosomes,can bind more ethidiumbromide because they canunwind as the ethidiumbromide binds. Linearmolecules can be saturatedwith ethidium bromide to thepoint of approximately onemolecule of ethidium bromidefor every two base pairs ofDNA. The ethidium saturatedDNA is centrifuged in a cesiumchloride gradient. Thedifferential binding of the dyeleads to different buoyantdensities for chromosomal andplasmid DNA in the cesiumchloride gradient. This allowsthe plasmid DNA to beseparated from thechromosome. While ethidiumbromide cesium chloridegradients are time consuming,for extremely pure plasmidDNA, they are still very 3/21/05 8:05 PM Page 234debris.

6 The supernatant contains a mixture of soluble cell components, including theplasmid, and is known as a methods used to purify the plasmid DNA from the cell lysate rely on the smallsize and abundance of the plasmid DNA relative to the chromosome, and the cova-lently closed circular nature of plasmid DNA. Most plasmids exist in the cytoplasm ofthe cell as circular DNA molecules that are highly supercoiled. The lysate is treatedwith sodium hydroxide to denature all of the DNA, and with detergent, SDS. The pHis then abruptly lowered, causing the SDS to precipitate and bring with it denaturedchromosomal DNA, membrane fragments, and other cell debris.

7 Most of the plasmidDNA renneals to form dsDNA because each strand is a covalently closed molecule andthe two strands are not physically separated from each other. The small size of theplasmid allows the plasmid molecules to remain in suspension. The supernatant,which contains plasmid DNA, proteins, and other small molecules can be treated in anumber of different ways to purify the plasmid. The most common protocol relies ona column resin that binds DNA. A small amount of the resin is mixed with the plas-mid-containing supernatant and the plasmid-bound resin is collected in a small col-umn.

8 The remaining cell components are washed away and the plasmid is elutedfrom the resin. This procedure is quick, simple, and reliable and can be easily carriedout on a large number of samples. Many modifications of this procedure have DNA isolationTo isolate chromosomal DNA, cells are lysed in much the same way as for plasmidDNA isolation. The cell lysate is extracted with phenol or otherwise treated to removeall of the proteins. The chromosomal DNA is precipitated as long threads. The chro-mosomal DNA is very fragile and breaks easily. For these reasons, the chromosomalDNA is not usually purified using columns.

9 Rather, the precipitated threads are col-lected by DNA moleculesOnce DNA has been purified, it must be cut into pieces before the chromosomal DNAand the plasmid DNA can be joined. The problem is to cut the DNA so that it will beeasy to join the cut ends of the chromosomal DNA to the cut ends of the plasmid group of enzymes, calledrestriction enzymes, are used for this purpose. Restric-tion enzymes are isolated from different bacterial use restriction enzymes and modification enzymes to identify their ownDNA from any foreign DNA that enters their cytoplasm. The restriction part of thesystem is an enzyme that recognizes a specific DNA sequence or restriction siteandcleaves the DNA by catalyzing breaks in specific phosphodiester bonds.

10 The cleavageis on both strands of the DNA so that a double-stranded break is made. The modifica-tion part of the system is a protein that recognizes the same DNA sequence as the re-striction enzyme. The modification enzyme methylates the DNA sequence so that therestriction enzyme no longer recognizes the sequence. Thus, the bacteria can protectits own DNA from the restriction enzyme. Any DNA that enters the bacteria and con-tains the unmethlyated restriction site is cut and degraded. There are three types of re-striction modification systems (Table ). The types are distinguished based on theDNA Cloning235 FYI discovery ofrestriction enzymesRestriction enzymes werediscovered in E.