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Lecture 5

MBB 222 Lecture Notes - Lecture 5: Polymerase Chain Reaction, Taq Polymerase, Cloning Vector


Department
Molec Biol & Biochem
Course Code
MBB 222
Professor
Edgar Young
Lecture
5

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Unit 4
Duplex stability
Duplex/double-stranded vs single stranded
o DNA is a double stranded molecule
o RNA is single stranded
DNA hybridization/annealing/renaturation vs denaturation/melting
o Annealing is the process of forming a DNA duplex from two single strands of DNA or a
single strand of DNA and a single strand of RNA
o Denaturation is the process of breaking the hydrogen bonds by applying heat to form a
partially denatured strand or completely denatured strand
RNA-DNA hybrid
o A hybrid double stranded molecule, consisting of a single strand of RNA and a single
strand of DNA
Exocyclic group
o A functional group located on the outside of a ring. Usually amino groups
Orbital hybridization (sp2 vs sp3)
o The mixing of orbitals during covalent bonding
Hydrogen bond acceptor vs hydrogen bond donor
o Hydrogen bond acceptors can accept hydrogen bonds, hydrogen bond donors can
donate hydrogen bonds with N, O, or F
H-bond directionality
o Planarity makes base stacking planar and straight. The pyrimidines are planar molecules
Conjugation/delocalization
o Movement of electrons that decreases the overall energy of a system, and increases
stability of it
P orbitals/pi bonds
Unshared pair
o An unshared pair of electrons can be present on atoms
Resonance hybrid vs resonance contributor
o A molecule can have multiple resonance hybrids in which some can have a full positive
or negative charge which may not be completely stable
o A resonance contributor is a resonance hybrid in which the contributor is the species in
which that molecule is found
o All nucleobases are conjugated systems with resonance structures which make them
suitable for long term information storage
Partial double-bond character
o Similar to resonance
o The participating bonds in a resonance hybrid have partial double-bond character, and
partial single bond character
Aromatic
o 4n+2 rule
o Has to be a closed ring
Tautomeric forms of nucleobases
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o Usually involves the movement of a hydrogen to another atom
o A real reaction with the addition of a proton (H+) and removal at two or more atoms
Melting temperature
o The melting temperature is the temperature at which DNA is denatured
o DNA containing more G-C pairs denatures at a higher temperature because G-C have
three hydrogen bonds *Remember, denaturation is breaking hydrogen bonds with
application of heat*
o So the melting temperature can suggest the composition of DNA
o Some A-T rich sequences will denature first, while the rest of the DNA is double
stranded
GC vs AT rich sequences
o DNA containing more G-C pairs denatures at a higher temperature because G-C have
three hydrogen bonds *Remember, denaturation is breaking hydrogen bonds with
application of heat*
o So the melting temperature can suggest the composition of DNA
o Some A-T rich sequences will denature first, while the rest of the DNA is double
stranded
UV light absorption at 260 nm
o Used to observe the transition from annealed to denatured DNA
Hypochromic effect vs hyperchromic effect
o Hypochromic effect
Close interaction between stacked bases in a nucleic acid decreases absorption
of UV light relative to that of a solution with the same concentration of free
nucleotides, and the absorption is decreased further when two complementary
nucleic acid strands are paired together.
o Hyperchromic effect
Denaturation of a double-stranded nucleic acid produces the opposite result: an
increase in absorption called the hyperchromic effect. The transition from
double stranded DNA to the single-stranded, denatured form can thus be
detected by monitoring UV absorption at 260 nm
DNA sequencing
Describe dideoxy(Sanger) dye-terminator method of DNA sequencing
DNA sequencing
Base-speicific reaction
Nucleotide analog
Dideoxynucleotide/ ddNTP (sanger method)
Dye-terminator
Labeling (fluorescent)
Polyacrylamide/capillary
o Polyacrylamide is the gel used in the matrix for short DNA molecules (up to a few
hundred nucleotides).
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o Agarose is generally used for longer pieces of DNA
Technique for PCR and Forensics
Describe polymerase chain reaction and its use in STR (short tandem repeat) genotyping
Polymerase chain reaction (PCR)
o Requires the sequence of the end portions of a DNA segment to be known
o Requires four components: DNA polymerase, dNTPs, a synthetic oligonucleotide primer,
and the DNA sample containing the segment to be amplified
Amplification
o The repeated cycle of heating is continued for about 25-30 minutes where primers
anneal and amplify the targeted sequence
Heating cycle
o A cycle of heating so the DNA strands denature so the primers can anneal to the strands
Heat-stable protein
o A protein that can resist denaturation from the heat
Taq DNA polymerase
o A polymerase that is derived from Taq bacteria and it is heat-stable
Mutagenic primer
RT-PCR
o After the DNA strand is made from the RNA template, the remaining cycles can be
carried out with DNA polymerases, using standard PCR protocols.
o Can be used to detect sequences derived from living cells as apposed to dead tissues
Reverse transcriptase
o An enzyme that works like DNA polymerase, but uses RNA as a template.
DNA genotyping (fingerprinting/profiling)
o A method based on sequence polymorphisms
Sequence polymorphism
o Slight sequence differences in individuals
Prototype sequence vs allele
Short tandem repeat (STR)
o A short DNA sequence, repeated many times in tandem at a specific location on a
chromosome; usually 4 bp long.
Dye-labeled primer
CODIS database
o Combined DNA Index System is based on 13 well-studied STR loci, which must be
present in any DNA-typing experiment carried out in the United States
DNA cloning
Describe a typical DNA cloning workflow (from DNA fragment to completed plasmid with amplification
in a bacterial host
Clone vs cloning/genetic engineering/ recombinant DNA
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