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| Lecture 2 - Nucleic Acid Biochemistry |
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Revised at 11:43 AM
Thursday, August 23, 2001
- Updated Fall 2001 material
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Chemical synthesis of DNA and RNA
Custom-designed oligonucleotides are available commercially and are used routinely in numerous experimental procedures. For example, oligonucleotides are used as template primers in DNA sequencing and PCR reactions, and for the incorporation of site-specific mutations in cloned genes. In addition, chemically-modified ribonucleotides can be used to synthesize large quantities of RNA for use as “antisense” or interference RNA (RNAi) inhibitors of RNA function.
In vitro DNA synthesis reactions take place inside sealed columns that contain glass beads which serve as the solid support for the sequential chemical reactions. Single phosphoramidites for each of the four bases are added to a growing chain that is initiated at the 3’ end.
The five chemical steps required for each nucleotide addition are;
1) De-blocking the 5’ end by DMT removal
2) Activation of the incoming phosphoramidite
3) Coupling of the nucleotides through a 5’-3’ linkage
4) Capping of unreacted nucleosides to prevent extension of incomplete products
5) Oxidizing the phosphate triester to stabilize the 5’-3’ linkage
What explains the observation that oligonucleotides of 20 bases or longer can initiate DNA synthesis at sites in a genome that are complementary to only 6-8 nucleotides in the primer under physiological conditions (37 degrees and 100 mM NaCl)?
What could you do to increase specifity in the reaction, e.g., to get the primer to work for PCR?
Why is high throughput oligonucleotide synthesis so important to applied molecular genetic methods, i.e., what is it about a template primer that makes it so useful?
DNA Metabolizing Enzymes
Restriction enzymes are the molecular scissors of molecular genetics, DNA ligase is the molecular glue, and DNA/RNA polymerases are the photocopying machines of the cell.
Restriction enzymes were discovered by microbiologists who were studying the host range of infectious bacteriophage. The term restriction refers to the fact that different strains of bacteria are resistant to bacteriophage infection if they contain the appropriate endonucleases that can destroy infecting DNA.
The bacteria protects its own genome from degradation by expressing site-specific methlyases that modify the host genomic DNA at the corresponding sequence specific sites in DNA. EcoRV is an example of a Type II restriction enzyme.
Restriction enzymes bind specific DNA sequences as dimers and catalyze the hydrolysis of the phosphodiester backbone on both strands of the DNA.
EcoRV cleaves at the recognition sequence: GAT ' ATC
BamHI cleaves at the recognition sequence: G ' GATCC
| Structure of BamHI dimer at non-specific site |
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| Structure of BamHI dimer at a specific site |
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Type II restriction enzymes cleave DNA in different ways and produce cohesive (complemenary) termini that can be used to create novel DNA molecules. Three types of termini can be generated; 1) 5' staggered ends, 2) blunt ends, and 3) 3' staggered ends. Importantly, the 5' termini of each strand in the cleavage product(s) retain the phosphoryl group from the phosphodiester bond, the 3' termini are hydroxylated.
Restriction enzyme recognition sites are often palindromic sequences as shown below for EcoRI, EcoRV and SacI.
What is the difference in the BamHI protein-DNA structure in the non-specific (scanning) conformation and specific site (cleavage) conformation?
What exactly is a palindrome? How is the palindromic sequence of a restriction enzyme recognition site related to the conformation (arrangement) of a restriction enzyme-DNA complex?
What is the recognition site sequence for the enzyme EcoRI methylase? Could bacterial strains contain only one of the two enzymes in a host restriction system, or are both enzymes (restriction enzyme and methylase) always required for the bacterial cell to survive?
DNA ligases are used to catalyze covalent bond formation between two DNA molecules. Bacteriophage T4 DNA ligase uses ATP as the phosphate donor as shown below. DNA ligases catalyze the formation of 5’-3’ phosphodiester bonds in double strand DNA molecules. In vivo, ligases function in DNA replication to repair single strand nicks resulting from DNA repair processes, and to join adjacent Okasaki DNA fragments produced in the lagging strand of a DNA replication fork. DNA ligase is used in recombinant DNA methods to covalently linked two heterologous DNA fragments as shown below.
What contributes to the low efficiency of blunt end ligations as compared to staggered end ligations?
Is it possible to ligate a DNA fragment cleaved with BamHI to a heterologous fragment that had been cleaved with the restriction enzyme Bgl2? (Hint: use the appendix in the textbook to determine the recognition site sequence of Bgl2).
When used as tools in recombinant DNA methods, restriction enzymes are more efficient at cleaving DNA into fragments, than is ligase in covalently linking together DNA fragments. What is the difference between the substrates in these two reactions, i.e., what is rate-limiting in the ligation reaction?
DNA and RNA polymerases direct the synthesis of complementary nucleic acids using single strand DNA as the template. DNA synthesis requires a preexisting DNA or RNA primer with a 3’ hydroxyl, whereas RNA synthesis can initiate synthesis without a primer. The in vitro enzymatic synthesis of DNA and RNA has become a central component in a variety of molecular genetic applications, for example, the amplification of DNA sequences by an enzyme called Taq DNA polymerase is used in the polymerase chain reaction (PCR).
Biochemical Methods Used to Analyze Nucleic Acids
One method used to biochemically purify supercoiled plasmid DNA is to physically separate different forms of the DNA on the basis of bouyant density in a Cesium Chloride equilibrium gradient in the presence of ethidium bromide.
Horizontal agarose gel electrophoresis is a common method used to analyze DNA fragments.
Dideoxy DNA sequencing is an enzymatic reaction used to decipher the nucleotide sequence of previously cloned DNA. In its simplest form (manual sequencing), this method is based on using four different chain termination nucleotides (ddATP, ddGTP, ddCTP, ddTTP) in separate reactions to generate DNA products that are resolved by polyacrylamide gel electrophoresis. Automated dideoxy DNA sequencing using fluourescently labled ddNTPS (chapter 9).
Why don't the chain-terminated products of the Sanger sequencing reaction all stop at the first nucleotide replacement, i.e., how is the "ladder" generated in the sequencing gel?
What is the purpose of adding 33P-dATP to all of the reactions? Could 33P-dCTP be used?
High throughput automated DNA sequencing uses one lane per template reaction, how is that possible considering that four different dideoxy dNTPs need to be used?
Southern Blots are used to identify complementary DNA sequences on the basis of molecular weight as analyzed using electrophoretic mobility in an agarose gel. Northern Blots are a variation of Southern Blots with the difference being that denatured RNA is electrophoresed through the agarose rather than DNA.
Southern Blot Procedure
Northern Blot Procedure
How did the name "Southern Blot" originate?
What is the purpose of soaking the agarose gel in NaOH prior to transferring the DNA to the filter membrane in a Southern Blot?
Why isn't renaturation in the high salt buffer (10xSSC) a problem during the transfer process?
How can you determine the molecular weight of a DNA fragment corresponding to a radioactive band on the film, if you do not use radioactive molecular weight markers?
Why is formaldehyde used to denature RNA in a Northern blot, isn't RNA single stranded to begin with? Why can't NaOH be used to denature RNA in the Northern blotting procedure as it is in the Southern Blotting procedure?
