EXAM 2. REVIEW III1. Chain termination (Dideoxyrebose) method2. High throughput method; Ppi, luciferase, adenosine 5’-phosphosulfate3. Stability of the DNA double helix4. ABZ DNA5. Intercalating agent6. DNA denatured/renatured7. Protein, DNA, RNA separation8. Plasmid, directional cloning9. Yeast Two Hybrid10. PCR11. Southern blot

11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids?

Figure 11.2 Primed synthesis of a DNA template by DNA polymerase, using the four deoxynucleoside triphosphates as the substrates.

11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids?

• DNA is a double-helical molecule • Each strand of the helix must be copied in

complementary fashion by DNA polymerase • Each strand is a template for copying • DNA polymerase requires template and primer • Primer: an oligonucleotide that pairs with the

end of the template molecule to form dsDNA • DNA polymerases add nucleotides in 5'-3'

direction

Chain Termination Method

• A template DNA with a complementary primer is copied by DNA polymerase in the presence of dATP, dCTP, dGTP, dTTP

• Solution contains small amounts of the four dideoxynucleotide analogs of these substrates, each of which contains a distinctive fluorescent tag, illustrated here as:

• Orange for ddATP• Blue for ddCTP• Green for ddGTP• Red for ddTTP

Occasional incorporation of a dideoxynucleotide terminates further synthesis of that strand

Figure 11.3 The chain termination method of DNA sequencing.

The set of terminated strands can be separated by capillary electrophoresis

High-Throughput DNA Sequencing by the Light of Fireflies

DNA polymerase produces PPi

ATP sulfurylase: PPi + APS → ATP + SO42-

Luciferase: ATP + luciferin + O2 → AMP + PPi + CO2 + oxyluciferin + light

Structures of luciferin and oxyluciferin. Light detection confirms that addition of a dNMP by primed synthesis has occurred.

High-Throughput DNA Sequencing by the Light of Fireflies

11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt?

• The stability of the DNA double helix is due to:• Hydrogen bonds – between base pairs• Electrostatic interactions – mutual repulsion of

phosphate groups, which makes them most stable on the helix exterior

• Base pair stacking interactions• Right-twist closes the gaps between base pairs to 3.4 A

(0.34 nm) in B-DNA• See Figure 11.6 for details of DNA secondary structure

Comparison of A, B, Z DNA

Intercalating Agents Distort the Double Helix

• The double helix is a very dynamic structure• Because it is flexible, aromatic macrocycles – flat

hydrophobic molecules composed of fused, heterocyclic rings, can slip between the stacked pairs of bases

• The bases are force apart to accommodate these intercalating agents– Ethidium bromide– Acridine orange– Actinomycin D

Intercalating Agents Distort the Double Helix

Figure 11.12 The structures of ethidium bromide, acridine orange, and actinomycin D, three intercalating agents, and their effects on DNA structure.

11.3 Can the Secondary Structure of DNA Be Denatured and Renatured?

See Figure 11.18• When DNA is heated to 80+ degrees Celsius, its UV

absorbance increases by 30-40% • This hyperchromic shift reflects the unwinding of

the DNA double helix • Stacked base pairs in native DNA absorb less light • When T is lowered, the absorbance drops, reflecting

the re-establishment of stacking

The Buoyant Density of DNA

Density gradient ultracentrifugation is a useful way to separate and purify nucleic acids.

The net movement of solute particles in an ultracentrifuge is the result of two processes: diffusion (from regions of higher concentration to regions of lower concentration) and sedimentation due to centrifugal force.

Telomeres and Tumors• The ends of chromosomes have specialized structures

known as telomeres - short, tandemly repeated nucleotide sequences at the ends of the chromosomal DNA

• Telomeres are added to the ends of chromosomal DNA by an RNA-containing enzyme known as telomerase

• Most normal somatic cells lack telomerase; thus with every cycle of cell division about 50 nucleotides are lost from each telomere

• The “telomere theory of aging” argues that telomere shortening is a factor in cell, tissue and organism aging.

Plasmids Are Very Useful in Cloning Genes

• Plamids are naturally occurring extrachromosomal DNA

• Plasmids are circular dsDNA • Plasmids can be cleaved by restriction

enzymes, leaving sticky ends • Artificial plasmids can be constructed by

linking new DNA fragments to the sticky ends of plasmid

• These recombinant molecules can be autonomously replicated, and hence propagated

Cloning VectorsCloning vectors are plasmids that can be modified to

carry new genes

• Plasmids useful as cloning vectors must have– a replicator (origin of replication)– a selectable marker (antibiotic resistance gene)– a cloning site (site where insertion of foreign

DNA will not disrupt replication or inactivate essential markers)

Directional CloningOften one desires to insert foreign DNA in a particular

orientation • This can be done by making two cleavages with two

different restriction enzymes • Construct foreign DNA with same two restriction

enzymes • Foreign DNA can only be inserted in one direction • See Figure 12.4

Directional Cloning

Figure 12.4 Directional cloning. DNA molecules whose ends have different overhangs can be used to form chimeric constructs in which the foreign DNA can enter the plasmid in only one orientation.

12.2 What Is a DNA Library?

A DNA library is a set of cloned DNA fragments that together represent the genes of a particular

organism • Any particular gene may represent a tiny, tiny

fraction of the DNA in a given cell • Can't isolate it directly • Trick is to find the fragment or fragments in the

library that contain the desired gene

cDNA Libraries Are DNA Libraries Prepared from mRNA

• cDNAs are DNAs copied from mRNA templates.• cDNA libraries are constructed by synthesizing cDNA

from purified cellular mRNA.• Because most eukaryotic mRNAs carry 3'-poly(A) tails,

mRNA can be selectively isolated from preparations of total cellular RNA by oligo(dT)-cellulose chromatography (Figure 12.9)

• DNA copies of the purified mRNAs are synthesized by first annealing short oligo(dT) chains to the poly(A) tails.

• These serve as primers for reverse transcriptase-driven synthesis of DNA (Figure 12.10)

Identifying Specific DNA Sequences by Southern Blotting

The Southern blotting technique involves the transfer of electrophoretically separated DNA fragments to an absorbent sheet and subsequent detection of the specific DNA sequences.

Specific Protein-Protein Interactions Can Be Identified Using the Two-Hybrid System

Figure 12.17 The yeast two-hybrid system for identifying protein-protein interactions. If proteins X and Y interact, the lacZ reporter gene is expressed. Cells expressing lacZ exhibit β-galactosidase activity.

12.4 What Is the Polymerase Chain Reaction (PCR)?

What if you don't have enough DNA for colony hybridization or Southern blots?

• The small sample of DNA serves as template for DNA polymerase

• Make complementary primers • Add primers in more than 1000-fold excess • Heat to make ssDNA, then cool • Run DNA polymerase (usually Taq) • Repeat heating, cooling, polymerase cycle

In Vitro MutagenesisFigure 12.19 One method of PCR-

based site-directed mutagenesis.

(1) Template DNA strands are separate and amplified by PCR.

(2) Following many cycles of PCR, the DNA product can be used to transform E. coli cells.

(3) The plasmid DNA can be isolated and screened for the presence of the unique restriction site (by restriction endonuclease cleavage.

Telomere, siRNA