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DNA
Coiling:
http://www.biostudio.
com/demo_freeman_
dna_coiling.htm
Replication: DNADNA
• Occurs during S phase
of mitosis in
reproducing cells only
• DNA template is copied
complimentarily and
semi conservatively
• Multiple oris (in
eukaryotes) and
replication forks form in
both directions,
producing a leading and
lagging strand as DNA
is elongated to the 3’
end
Complimentary & Antiparallel
• DNA end with the
phosphate attached to
the #5 carbon = 5’ end
• DNA end with the OH
group attached to the #3
carbon is the 3’end
• Synthesis of a n.a.
chain always proceeds
5’ to 3’
Okazaki Fragments
• Fragments of DNA 10002000bp long
• Produced on the lagging
strand
• Must be joined with
ligase
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DNA Replication Flash: http://207.207.4.198/pub/flash/24/menu.swf
DNA Replication: http://www.ncc.gmu.edu/dna/repanim.htm
Replication:
http://nobelprize.org/educational_games/medicine/dna/a/replication/replication_a
ni.html
DNA Replication Fork & Nucleotide Addition: http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter14/animations.html#
Enzymes
• DNA polymerase adds nucleotides, lengthens DNA
(Also proofreads sequence and undoes 10% of bases )
• Ligasejoins nucleic acid pieces
• Primase joins RNA primer to DNA strand
• Helicaseunwind the DNA
• Nuclease cuts DNA
• Endonuclease cuts DNA internally
• Telomerasecatalyzes lengthening of telomeres
Gene processing
Mistakes/Mutations
• Mutation any permanent
alteration in the sequence
of DNA bases
• Point mutations can result
in frameshifts in the
reading frame
• Chromosomal mutations
can result in translocations
Chromosomal Mutations
Base-pairing (substitution/
deletion-addition) Mutations
Mistakes/Mutations
• Silent mutation
change in nt sequence,
but no change in
product
• Nonsense mutation
insertion of stop codon
into sequence
• Missense mutation
swapping of aa (sickle
cell anemia, PKU)
Types of DNA
Coding DNA
Less than 5% of the human
genome actually codes
for genes
Simple Sequence DNA
20-30% human DNA
Short sequence of 5-10 bp repeat.
Half of the DNA in a species of crab has
ATATATATA
Fruit fly has ACAAACT 12 million times
Intermediate-Repeat
150-300bp scattered throughout genome, some have
known functions and code for histones and rRNA
Huntington’s disease CAG repeats 6-35/38-180bp (also
various dystrophies)
Junk DNA ?
More than 95 percent of all DNA, was called "Junk
DNA" by molecular biologists, because they were
unable to ascribe any function to it. However that
category is becoming smaller as scientists identify
non-coding DNA as introns, transposable
elements, pseudogenes, VNTRs (variable number
tandem repeats and “junk”.
Extraneous DNA
proviruswhen the DNA
of a virus incorporates its
self into an eukaryotic
cell. Can move around in
the genome
reverse transcriptase
enzyme that allows RNA
strand to be a template of
DNA
transposons “jumping
genes”
Where did it come from?
One hypothesis about the junk is that these chromosomal
regions are trash heaps of defunct genes, sometimes known
as pseudogenes, which have been cast aside and fragmented
during evolution.
Evidence for a related hypothesis suggests that the junk
represents the accumulated DNA of failed viruses.
Yet another hypothesis is that the junk DNA provides a
reservoir of sequences from which potentially advantageous
new genes can emerge. (Junk vs trash)
As with most things in science, time and technology will
tell…
Types of RNA
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mRNA “messenger RNA” Transcribe DNA to RNA
rRNA “ribosomal RNA’ used in the building of ribosomes
tRNA “transfer RNA” carry a.a. to ribosome during translation
snRNA “small nuclear RNA” Mediate the processing of DNA
transcripts for mRNA, rRNA, and tRNA
snoRNA “small nucleolar RNA”RNAs within the nucleolus have
several functions
miRNA“micro RNA” tiny (~22 nts) RNA molecules that appear to
regulate the expression of mRNA
XIST RNA. inactivate one of the two X chromosomes in female
vertebrates.
gRNA “guide RNA” found in mitochondrial DNA
sl RNA “splice leader RNA” 5’mRNA cap
hnRNA “heterogenous nuclear RNA” nonspliced or immature
RNA
Isolating DNA
Restriction enzymes/
endonucleases cut at
specific palindromic
sequences
5’
3’
G/AATT C
C TTAA/G
3’
5’
cleavage leaves “sticky ends”
G
AATTC
CTTAA
G
Steps in Cloning a Gene
DNA Restriction Step by Step
RFLP Analysis
• Restriction Fragments. Those are the fragments of DNA that were cut
by restriction enzymes.
• Length, and refers to the length of the restriction fragments.
• Polymorphism, a Greek term that literally means "many shapes". The
lengths of some of the restriction fragments differ greatly between
individuals, thus there are many shapes, or lengths, of DNA possible.
• Molecular biologists have identified regions of the human genome
where restriction fragment lengths are highly variable between
individuals. These regions are called RFLP markers.
VNTR Tutorial
Electrophoresis
• Move DNA through a
matrix with a sieving
effect
• Agarose gels for DNA
• PAGE for proteins
• Single digest uses 1
enzyme
• Double digest produces 2
restriction patterns
Interactive Animation, Simplified
Blotting
• Goes from the gel to filter paper to
make and autoradiograph
• Southern blot: uses RNA to probe DNA
strands (SB2)
• Northern blot: uses DNA to probe RNA
strands
• Western blot: “immunoblotting” uses
pp-antibody binding reaction
Hybridization
• These hybridization
reactions can be used
to detect and
characterize
nucleotide sequences
using a particular
nucleotide sequence
as a probe.
• used to find
relatedness in n.a.
Polymerase Chain Reaction (PCR)
PCR2 PCR3 PCR4 PCR Rap PCR Song
• Construction of a Plasmid Vector
Transcription: DNAmRNA
• Occurs continually
throughout the cell
cycle
• Occurs in the
nucleus as
messenger RNA
copies DNA
• cDNA
• Operon=promoter + operator + TFs
• Promoter region, upstream of gene, signals start of
transcription
• Transcription factors mediate binding of RNA polymerase
• Operon is controlled by regulatory genes such as repressor
genes
Control in Prokaryotes: http://trc.ucdavis.edu/biosci10v/bis10v/week6/08lacoperon.html
Transcription
Initiation Complex
Splicing of introns & exons
• Non-coding regions
(introns) of mRNA are
spliced out of the mRNA
strand before leaving the
nucleus
• 5’cap is methylated and
3’tail is polyadenylated
to indicate orientation at
the ribosome
Translation: mRNAprotein
• Aamino acyl (initiation with Met.)
• Ppeptide bond (elongation)
• Eexit (termination with UAG, UAA, UGA)
Regulatory Proteins
• Lac Operon
• Lac Operon Induction
• Regulatory Proteins: Repression
Signal Transduction
• Signal Transduction Pathway
• My Dog is Broken (A Case Study for Cell
Signaling)
GMOs
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Classical vs Transgenic Breeding
Harvest of Fear (What about this fish?)
Babies by Design?
Transgenic Mouse
Resources
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BioStudio Animations: http://www.biostudio.com/a_sitemap.htm
Biology Animations: http://science.nhmccd.edu/biol/ap1int.htm
Restriction Mapping Tutorial:
DNA Replication:
BioZone BioLinks: http://www.biozone.co.nz/CELL_BIOL_AND_BIOCHEM.html
Lac Operon Animation:
Lac Operon Induction:
Biointeractive Animations:
Animated Tutorials: Molecular Biology:
Processing of Gene Information Animation Prokaryotes vs Eukaryotes:
Learner’s TV:
Fundamentals of Molecular Diagnostic Links:
Gene Expression
DNA Technology Quiz