Presentation on theme: "How do we analyze DNA? Gel electrophoresis Restriction digestion"— Presentation transcript:
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 improveBUT - 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 Electrophoresis1949 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 indispensableWide 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 evidenceMen proving or disproving paternity via the techniqueHospitals 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.
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 enzymesGel 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 nucleotidesThese smaller pieces can then be cloned into a plasmid or bacteriophage vector for amplification and further analysis
9 Restriction MappingA restriction map is a description of restriction endonuclease cleavage sites within a single piece of DNAFirst step in characterizing an unknown DNA, and a prerequisite to manipulating it for other purposesRestriction 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 Digest samples of the plasmid with a set of individual enzymes, and with pairs of those enzymesDigests are then "run out" on an agarose gel to determine sizesDeduce 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)
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 SequencingShort stretches of DNA can be sequenced using “primers” that are based on the known nucleotides where the restriction enzyme has cut the DNADNA heated to a critical temperature called the Tm will “denature” or separate into two single strandsThese strands can be used to build new complementary strands, or can “reanneal” or stick back together when the temperature is reduced
14 Polymerase Chain Reaction (PCR) 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.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.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'
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 tagAs the strand is replicated it will stop elongating each time one of these dideoxynucleotides is added
17 DNA SequencingAs 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
18 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 anotherDNA fingerprints allow for identification of each individualOnce genes have been identified we can begin to work with these areas of the genome in order to improve traits