Biochemistry Topics
Restriction Endonucleases
Restriction endonucleases recognize a specific nucleotide sequence in double stranded DNA to be cleaved. Some restriction endonucleases cleave in different places at each DNA strand leaving "sticky ends". Their biological role is to cleave foreing DNA. The cell's own DNA is not degraded because the sites recognized by its own restriction endonucleases are methylated. Cleavege sites have two-fold symetry: the recognized sequence is palindromic and the cleavage sites at each strand are symetrical.
Restriction endonucleases are used to cleave DNA molecules into specific fragments that are more easily analyzed and manipulated than the parent molecule. Small differences between related DNA molecules can be readily detected because their restriction fragments can be separated and displayed by gel electrophoresis.
Take Quiz: [Q1] [Q2] [Q3]
Advance Topics: Cellular
and Molecular Biology
Molecular Biology Lab
DNA Sequencing
Two methods have been developed to sequence DNA: the Maxam-Gilbert chemical method and the Sanger enzymatic method.
Maxam-Gilbert is a chemical cleavage method that starts with single DNA radiolabeled labeled at one end with (32)P. The labeled DNA is then broken preferentially at one of the four nucleotides. Conditions are chosen so that an average of one break is made per chain. In the reaction mixture for a given base, each broken chain yields a radioactive fragment extending from (32)P to one the positions of the cleavage base. Such fragments are produced for every position of the chosen base. The same reaction is done for each of the bases and each reaction mixture is separated by polyacrylamide gel electrophoresis. The gel will resolve DNA fragments difering in length by only one nucleotide. The autoradiography of the gel displays a pattern of bands from which the sequence can be read directly.
The controlled interruption of enzymatic replication developed by Sanger is now the method of choicebecause of its simplicity. DNA polymerase is used to copy a particular sequence of single-stranded DNA using radiolabeled deoxynucleotides (dNTPs) and a 2'-3'-dideoxy analog of one nucleotide. When the 2'-3'ddNTP is used to synthetize a base pair, it blocks further grow of the new chain because it lacks the 3' hydroxy terminus needed to form the next phosphodiester bond. Hence fragments of various lengths are produced. Four such sets of reactions, one for each dNTP, are electrophoresed and the base sequence read from the autoradiogram.
Take Quiz: [Q1] [Q2] [Q3]
Cloning Vectors
Topic not yet available
Take Quiz: [Q1] [Q2] [Q3]
Genomic and cDNA Libraries
Topic not yet available
Take Quiz: [Q1] [Q2] [Q3]
PCR
Topic not yet available
Take Quiz: [Q1] [Q2] [Q3]
Continue
to "Final Exam" or take a test: [T1] [T2] [T3].
Need more practice? Answer the following review questions:
Questions not yet available