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Chapter 11

BIOCHEM 2B03 Chapter Notes - Chapter 11: Interphase, Thymidine Triphosphate, Dna Supercoil


Department
Biochemistry
Course Code
BIOCHEM 2B03
Professor
Margaret Fahnestock
Chapter
11

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Biochem 2B03
Jasmyn Lee
1
Chapter 11: Structure of Nucleic Acid
11.1 How Do Scientists Determine The Primary Structure of Nucleic Acids?
The nucleotide sequence of DNA can be determined from the electrophoretic migration of a defined set of
polynucleotide fragments
Chain termination/dideoxy method more common protocol for nucleic acid sequencing
o Fredrick Sanger
o Relies on enzymatic replication of the DNA to be sequenced
Sanger’s chain termination/dideoxy method uses DNA replication to generate a defined set of polynucleotide
fragments
Biochemistry of DNA Replication
o DNA polymerase copies the sequence of nucleotides in one strand in a complementary fashion to form
a new second strand
o Each original strand of the double helix serves as a template
o DNA polymerase carries this out in the presence of four deoxynucleotide monomers and copies single-
stranded DNA, provided a double-stranded region of DNA is artificially generated by adding a primer
Primer an oligonucleotide capable of forming a short stretch of dsDNA by base pairing with
the ssDNA
o Primer must have a free 3-OH end from which the new polynucleotide chain can grow as the first
residue is added in the initial step of the polymerization process
o DNA polymerase synthesize new strands by adding successive nucleotides in the 5’3’ direction
Chain Termination Protocol Sanger DNA sequence method
o Clones derived from PCR products need to be sequenced to rule out errors
o Sanger sequencing makes use of a short DNA primer and a single subunit DNA polymerase (derived
from T7 bacteriophage usually
o Primer requirement is met by an appropriate oligonucleotide
o Reaction run in the presence of
All four deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) which are the substrate for
DNA Polymerase
All four corresponding 2’,3’-dideoxynucleotides
o Depends on base-specific inhibitors
o 2’,3’-dideoxy nucleoside triphosphate lack 3’-OH groups and cannot serve as acceptors for 5’-nucleotide
addition in the polymerization reaction inhibits progression of DNA polymerase; chain is terminated
where they are incorporated

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Biochem 2B03
Jasmyn Lee
2
No nucleophile to attach the 5’ phosphate
o Fewer dideoxynucleotide than deoxynucleotides chain is terminated infrequently and randomly a
population of new strands of varying lengths is synthesized
o Add fluorescently labeled 2’3’ dideoxynucleoside triphosphates to a DNA synthesis reaction at ~10x
lower concentration than the 2’ deoxynucleoside triphosphates
1. Causes chain termination
2. Labels the terminated ends with a base-specific fluorescent label
o These can then be separated on the basis of length using capillary gel electrophoresis
o The order of different colored fluorescent markers allows you to read the DNA sequence directly
o Eg/ Gel read 5’TTGTCGAAGTCAG3’ sequence is complementary to template DNA strand
5’CTGACTTCGACAA3’
11.2 What sorts of Secondary Structures Can Double-Stranded DNA Molecules Adapt?
DNA usually occurs in the form of double stranded molecules
o DNA is a two chain structure with hydrogen bonds formed between opposing based on the two
antiparallel chains
o Polar sugar phosphate backbones on the outside; bases stacked on the inside (0.6 nm apart when in
ladder-like structure)
o Ladder-like structure converts into a double helix when given a right handed twist brings base pair
rungs closer together (0.34 nm apart) B-DNA
Watson-Crick base pairs (A:T pair and G:C pair) have virtually identical dimensions
DNA double helix is a stable structure
o H-Bonds
2 in A:T pair
3 in G:C pair
H bonds form between H2O and bases when strands are separated

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Biochem 2B03
Jasmyn Lee
3
Polar atoms in sugar-phosphate backbone form external H bonds with H2O
o Electrostatic Interactions
Negatively charged phosphate groups along the sugar-phosphate backbone keep strands
apart except when base pairing occurs
Charges become electrostatically shielded from one another because divalent cations (i.e. Mg2+)
bind strongly to the anionic phosphates
o Van der Waals and Hydrophobic Interactions
Base pairs stick together through π,π-electronic interactions and hydrophobic forces
Two glycosidic bonds holding the bases in each base pair are not directly across the helix from
each other
Sugar-phosphate backbones of the helix are nonequally spaced along the helix axis and
the grooved between them are not the same size major groove and minor groove
o The “tops” of the bases line the “floor of the major groove”
Top defined as placing the glycosidic bond at the bottom
o The “bottoms” of the bases line the floor of the minor groove
Some proteins that bind to DNA can recognize specific nucleotide sequences by reading
the pattern of H-bonding possibilities presented by the edges of the bases in these
grooves
Watson-Crick base pairs as they project into the grooves
o Chemical information in major groove: CG base pair NH2 O
GC base pair O NH2
AT base pair NH2 CH3
TA base pair CH3 NH2
o Chemical information in minor groove: GC base pair NH2 O
CG base pair O NH2
AT base pair O
TA base pair O
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