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pGLO lab
The importance of DNA
The DNA double helix is the code of
life
• DNA is the code to
make proteins
• The blueprint for all
structures in your
body which are
made of protein
• DNA is comprised of
nucleotides
Nulceotides are the monomers of
nucleic acid polymers
• Consist of a sugar, a
phosphate, and a
nitrogen-containing
base
• Sugar can be
deoxygenated
• Bases contain the
genetic information
There are 4 kinds of DNA bases
Adenine always
matches with
Thymine,
Cytosine always
matches with
GuanineHydrogen bonds
hold bases
together
Living things are extremely complex
• Cellular machinery is
sophisticated and required for
life
• Cellular machinery is made
largely of proteins
• Blueprints for all cellular
machinery are contained in
genes
• Genes are inherited from
parents
• Humans have ~30,000 genes
Proteins give living things the variety
of their structures
Protein variety is generated by 1o
structure- the sequence of amino acids
which make the protein
Amino Acids
• Proteins consist of subunits called amino acids
Figure 2.12
How DNA works
• Replication
• Transcription
• Translation
The sequence of DNA bases is the
code for the primary structure of
proteins
All cells require a copy of the genome
•
•
•
•
Genome- all the genes of the cell
Human genome is made of DNA
DNA is similar in all cells
Gene- 1 DNA Molecule (+
proteins the genetic information
to produce a single product
(protein)
• DNA replication copies all cellular
DNA
Replication of DNA
Figure 21.2
The DNA code
Computers use binary digital code
01000011 01101000
01100001 = A
01100101 01100101
01100010 =B
01110011 01100101
01000011 =c
01100010 01110101
01110010 01100111
00100111 = apostrophe
01100101 01110010
Etc.
00100000 01000100
01100101 01101100
• http://www.geek01110101 01111000
notes.com/tools/17/tex
01100101 =
t-to-binary-translator/
cheeseburger deluxe
•
•
•
•
•
How does the DNA code work?
• atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaa
gttcgtatggaaggttccgttaacggtcacgagttcgaaatcga
aggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgct
aaactgaaagttaccaaaggtggtccgctgccgttcgcttggga
catcctgtccccgcagttccagtacggttccaaagcttacgttaa
acacccggctgacatcccggactacctgaaactgtccttcccgg
aaggtttcaaatgggaacgtgttatgaacttcgaagacggtggt
gttgttaccgttacccaggactcctccctgcaagacggtgagttc
=GFP
The DNA
code is
(nearly)
universal
It uses groups of 3
bases (codon)
3 bases = 1 codon = 1
amino acid
The Central Dogma of Molecular
Biology
• DNA RNA Protein
• DNARNA :
Transcription
• RNA Protein:
Translation
DNA RNA Protein Trait
The
Universality
of the DNA
code makes
this
possible
Firefly gene (Luciferase) in a tobacco plant
tRNA’s carry an amino acid at one end, and
have an anticodon at the other
Amino acid
attachment site:
Binds to a specific
amino acid.
Amino acid
(phenylalanine)
Anticodon:
Binds to codon on mRNA,
following complementary
base-pairing rules.
Anticodon
mRNA
Figure 21.6
The ribosome matches tRNA’s to the
mRNA, thereby linking amino acids in
sequence
In this way, the proteins in nature are
virtually limitless
Proteins are incredibly diverse at the
molecular level
A few examples
Insulin
Rubisco
ATP synthase
Fibrin
Nitrogenase
Protein function depends greatly on shape
Because the DNA code is universal, genes can be moved
from one living thing to another
Cell with gene of interest
Bacterium
Step 1: Isolate DNA from
two sources.
Step 2: Cut both DNAs
with the same
restriction enzyme.
Plasmid
Source (donor) DNA
Fragments of
source DNA
Step 3: When mixed, the
DNAs recombine by base
pairing.
Figure 21.14 (1 of 2)
When one DNA molecule is copied to make two
DNA molecules, the new DNA contains
1.
2.
3.
4.
5.
A) 25% of the parent DNA.
B) 50% of the parent DNA.
C) 75% of the parent DNA.
D) 100% of the parent DNA.
E) none of the parent DNA.
DNA in action
• HIV and AIDS
• Genetic engineering
• pGLO
Genetic
Engineering
allows DNA
to be moved
from one
organism to
another
Figure 21.14
Green Fluorescent Protein
•
discovered in 1960s by Dr. Frank Johnson
and colleagues
•
closely related to jellyfish aequorin
•
absorption max = 470nm
•
emission max = 508nm
•
238 amino acids, 27kDa
•
“beta can” conformation: 11 antiparallel
beta sheets, 4 alpha helices, and a
centered chromophore
•
amino acid substitutions result in several
variants, including YFP, BFP, and CFP
40 Å
30 Å
The pGLO plasmid has genes which can be
turned on and off
• ori- origin of replication
• GFP- green fluorescent
protein
• bla- Beta-lactamase
• araC- Arabinose
• What are all the other
marks? Why are they
there?
2008 Nobel Prize- GFP
• GFP mice
On pGLO, the regulatory regions of the
Arabinose operon have been glued to the
structural sequences for GFP
ara GFP Operon
ara Operon
B
ara
C
A D
araC
GFP Gene
Effector (Arabinose)
Effector (Arabinose)
Gene Regulation
B A D
araC
araC
GFP Gene
RNA Polymerase
RNA Polymerase
araC
B A D
araC
GFP Gene
What will happen on the Ara (+) plates?
What will happen on the Ara (-) plates?
Grow? Glow?
Which colonies will glow?



Follow protocol
On which plates will
colonies grow?
Which colonies will
glow?
5/23/2017
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