How do we analyze DNA?

1. How do we analyze DNA? • Gel electrophoresis • Restriction digestion • Sequencing

2. DNA analysis • How do we manipulate DNA to improve our crop? • First we need to identify which genes in the DNA sequence are important for the trait we are trying to improve • BUT - DNA from a single human cell can be over 2m in length! Some plants have genomes as large as 7.5 x 1010 base pairs (25 times the size of the human genome).

3. Gel Electrophoresis • 1949 a team led by chemist Linus Pauling found two samples of hemoglobin from healthy and sickle-cell anemia sufferers migrated at different rates. • Today, gel electrophoresis is indispensable • Wide variety of applications includes determination of a gene's sequence, isolation of entire chromosomes, and separation and characterization of proteins

4. Uses outside the laboratory • DNA fingerprinting evidence • Men proving or disproving paternity via the technique • Hospitals replacing conventional heel prints with genetic fingerprints as a means of identifying newborns.

5. How does it work? • Agarose and polyacrylamide are the two media most commonly used in gel electrophoresis. Both substances create a porous matrix through which charged macromolecules migrate in response to an electric field. • Negatively charged DNA, for example, travels toward the positively charged electrode when current is applied. http://www.life.uiuc.edu/molbio/geldigest/photo.html

6. Pores in the matrix limit the migration of large molecules, while smaller molecules migrate more freely and travel farther toward the opposite pole. This molecular sieving separates molecules on the basis of size. • Agarose is used as the support matrix to separate nucleic acids and very large proteins or complexes. • Agarose is a natural polysaccharide derived from certain types of red seaweed. When heated and then cooled, agarose solidifies into a solid matrix with relatively large, nonrestrictive pores.

7. Agarose gel electrophoresis (AGE) can be used to separate molecules by charge or by their molecular weight. • One of the most common applications of AGE is its use in separation of the fragments generated by cleaving DNA with restriction enzymes • Gel electrophoresis not only separates macromolecules, but also allows the researcher to actually use the nucleic acid or protein by transferring it to a support membrane made of nitrocellulose or nylon, and then probing it with radioisotope- or enzyme-labeled complementary DNA or antibodies

8. DNA has to be cut into more manageable pieces before it can be analyzed Restriction enzymes cut DNA in very specific places that are determined by the order of 4-8 base pairs of nucleotides These smaller pieces can then be cloned into a plasmid or bacteriophage vector for amplification and further analysis

9. Restriction Mapping • A restriction map is a description of restriction endonuclease cleavage sites within a single piece of DNA • First step in characterizing an unknown DNA, and a prerequisite to manipulating it for other purposes • Restriction enzymes that cleave DNA infrequently (e.g. those with 6 bp recognition sites) and are relatively inexpensive are used to produce a map

10. Creating a Map - Digestion with Multiple Restriction Enzymes Digestsamples of the plasmid with a set of individual enzymes, and with pairs of those enzymes Digests are then "run out" on an agarose gel to determine sizes Deduce where each enzyme cuts, which is what mapping is all about

11. e.g. Plasmid with 3000 base pair (bp) fragment of unknown DNA • Digestion with Kpn I yields two fragments: 1000 bp and "big“ • Digestion with BamH I yields 3 fragments: 600, 2200 and "big“ • double digest yields fragments of 600, 1000 and 1200 bp (plus the "big" fragment) http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/maps.html#top

12. If the process outlined above were conducted with a larger set of enzymes, a much more complete map would result. • In essence, single digests are used to determine which fragments are in the unknown DNA, and double digests to order and orient the fragments correctly.

13. DNA Sequencing • Short stretches of DNA can be sequenced using “primers” that are based on the known nucleotides where the restriction enzyme has cut the DNA • DNA heated to a critical temperature called the Tm will “denature” or separate into two single strands • These strands can be used to build new complementary strands, or can “reanneal” or stick back together when the temperature is reduced http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

14. Polymerase Chain Reaction (PCR) • For each strand, we provide a primer, which is a short piece of DNA that sticks to one end of the strand. • Any single-stranded piece of DNA can only hybridize with another if their sequences are complementary. If we have just one strand, we can actually build another strand to match it. http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html • DNA polymerase "reads" the bases on one strand and can attach the complementary base to the growing strand.

15. DNA sequencing reactions are just like the PCR reactions for replicating DNA • The reaction mix includes the template DNA, free nucleotides, an enzyme (usually a variant of Taq polymerase) and a 'primer' http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

16. A small proportion of each of the four bases in the reaction mixture is specially modified to form a dideoxynucleotide and is labeled with a unique fluorescent dye or tag • As the strand is replicated it will stop elongating each time one of these dideoxynucleotides is added http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

17. DNA Sequencing • As the reaction proceeds to build a new DNA strand from the existing one the signal from each of the bases can be recognized and so we can work out in which order the bases were added http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

18. http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.htmlhttp://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html • A 'Scan' of one gel lane: The computer “reads” the lane for us! This is what the sequencer's computer shows us - a plot of the colors detected in one 'lane' of a gel (one sample), scanned from smallest fragments to largest. The computer even interprets the colors by printing the nucleotide sequence across the top of the plot

19. How do we use this information? • We can compare organisms to one another • DNA fingerprints allow for identification of each individual • Once genes have been identified we can begin to work with these areas of the genome in order to improve traits