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IB HL 2.7 and 7.1 DNA Structure and Replication (Structure (Rosalind…
IB HL 2.7 and 7.1 DNA Structure and Replication
Replication
Semi-conservative: one strand will be from template molecule, one newly synthesized
Each nitrogenous base can only pair w/ complementary partner (Adenine and thymine, cytosine and guanine), therefore each new strand will be identical base sequence to original
Meselson-Stahl experiment: nitrogen radioisotopes used, DNA prepared using 15N, induced to replicate in 14N, samples then separated via centrifugation to determine composition (contained mix of N's after one divis., some contained only 14N after two divis.)
Three hypotheses
Conservative: new molecule synthesized from unaltered template
Semi-conservative: each new molecule consists of one newly synthesized and one template
Dispersive: new molecules made up of segments of new and old
Polymerase chain reaction: artificial method of DNA replication in lab; standard sequence creates over one billion copies
Steps
1) DEnaturation: DNA sample heated to 90C to separate strands
2) annealing: sample cooled to 55C, allowing primers to anneal (designating sequence to be copied)
3) elongation: sapmle heated to optimal temperature for heat-tolerant polymerase to function at 75C
Taq polymerase: enzyme isolated from thermophilic bacteria thermus aquaticus; extends nucleotide chain from primers, therefore primers used to select sequence to be copied
DNA Replication process
1)
helicase unwinds, breaks H-bonds
b/t b/p's to separate two strands; occurring at origins of replication and creating replication fork of two strands in antiparallel directions
2)
DNA gyrase reduces torsional strain
by relaxing positive supercoils that would form during unwinding
3)
single-stranded binding proteins binds to DNA
strands after separation and
prevent re-annealing
; also prevent single-stranded DNA from being digested by nucleases; dislodged when new complementary strand synthesized
4)
DNA primase generates short RNA primer
(10-15 nucleotides) on each of templates strands,
providing initiation point for DNA polymerase III
(it can extend chain but not start one)
5)
DNA polymerase III attaches 3'-end of primer and covalently joins free nucleotides in 5' to 3' direction
(after they align opposite complementary b/p); moves in opposite directions - on leading strand, moves towards replication fork, can synthesize continuously; on lagging strand, moves away from fork and synthesizes in Okazaki fragments
6)
DNA polymerase I removes RNA primers from lagging strands
, replacing them w/ DNA nucleotides (multiple primers along length as Okazaki fragments synthesized)
7)
DNA ligase joins Okazaki fragments
to form strand by covalently joining sugar-phosphate backbones w/ phosphodiester bond
DNA sequencing: process by which base order of nucleotide sequence is explained by chain-terminating dideoxynucleotides
ddNTPs lack 3' hydroxyl group necessary for phosphodiester bond therefore preventing further elongation of nucleotide chain; length of sequence will reflect position at which ddNTP incorporated
Sequencing via Sanger method: four PCR mixes set up, each containing stocks of normal nucleotides plus one ddA, ddT, ddC, or ddG dideoxynucleotide; when fragments separated via gel electrophoresis, base sequence determined by ordering fragments according to length
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DNA polymerase can only add nucleotides to existing strand therefore RNA primer must be synthesized for attachment point
Free nucleotides exist as dNTPs w/ 3 phosphate groups; polymerase cleaves additional 2 phosphates and uses energy to form phosphodiester bond w/ 3' end
On lagging strand, polymerase moving away therefore must constantly return to copy newly separated stretches, therefore copied as short Okazaki fragments as preceded by primer (eventually primers replaced w/ DNA bases and fragments joined together)
Structure
Hershey and Chase: scientists unsure as to whether DNA or protein was genetic material
Procedure: viruses grown in 1/2 isotopic mediums to radioactively label viral component (sulfur present in proteins, and phosphorus present in DNA); viruses allowed to infect bacterium, then separated via centrifugation, larger bacteria formed pellet, found to be radioactive when infected by DNA but not protein
Rosalind Franklin and Maurice Wilkins
Procedure: DNA purified, fibres stretched into thin glass tube, DNA targeted by x-ray beam which diffracted when contacted atom; pattern recorded and used to find details
Findings
Composition: double-stranded
Orientation: nitrogenous bases closely packed together on inside, phosphates form outer backbone
Shape: DNA twists at regular intervals to form helix
Watson and Crick
A-T bond (paired via 2 H-bonds) same length as G-C (paired via 3 H-bonds)
Replication occurs via complementary base pairing and is bidirectional due to antiparallel nature
Non-coding DNA
Examples
Satellite DNA: tandemly repeating sequences, structural component of heterchromatin, centromeres
Telomeres: protect against chromosomal deterioration
Introns: non-coding sequences WITHIN genes, removed by RNA splicing before mRNA formation
Non-coding RNA genes: codes for RNA molecules not translated into proteins
Gene regulatory sequences: involved in transcription (promoters, enhancers, silencers)
DNA profiling: individuals can be identified and compared
Non-coding regions there are satellite DNA
Tandem repeats excised using restriction enzymes, separated w/ gel electrophoresis
As individuals will likely have different number of repeats at given locus, they will generate unique profiles
Nucleosomes: DNA packaged with histone proteins in eukaryotes; help to supercoil DNA, protect it from damage, allows chromosome mobility
Organisation
DNA complexed with octamer
Negative DNA associates w/ positive amino acids on histone proteins
Histone proteins have N-terminal tails that extrude outwards from nucleosome; tails from adjacent histone octamers link up and draw nucleosomes close together during condensation
Nucleosomes linked by additional histone protein to form string of chromatosomes
Coil to form solenoid structure, condensed to form 30 nm fibre
Fibres form loops, compressed and folded around protein to form chromatin
Chromatin supercoils during cell division to form chromosomes