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From DNA
To RNA
To Protein
A Nucleotide
Phosphate
HO
OH
P
O
Base
NH2
N
O
CH2
O
N
Sugar
OH
H
OH
N
N
Two Families of Bases
Purines
NH2
Adenine
N
N
N
Pyrimidines
O
CH3
N
O
N
Guanine
NH
N
(DNA)
NH2
O
Uracil
(RNA)
NH
N
N
Thymine
O
NH
N
O
NH2
Cytosine
N
N
O
Hydrogen bonds
2 H- bonds for A:T
3 H-bonds for G:C
Ribose
Deoxyribose
Genomes vary in size
Introduction
The Central Dogma
of Molecular Biology
Cell
DNA
Transcription
Translation
mRNA
Ribosome
Polypeptide
(protein)
Transcription And Translation In
Prokaryotes
5’
3’
3’
5’
RNA
Pol.
Ribosome
mRNA
5’
Eukaryotic Transcription
Cytoplasm
Ribosome
DNA
Transcription
Translation
preRNA
RNA
Processing
mRNA G
G
AAAAAA
Nucleus
Export
AAAAAA
Nucleotide Words
• Words in the nucleotide language are all 3
letters or bases long.
• These three base “words” are called codons
• This means that there can only be 43 = 64
unique words.
A Codon
OH
P
HO
NH2
O
N
O
CH2
N
O
P
O
O
N
O
CH2
P
NH
N
O
Guanine
NH2
N
H
O
HO
N
H
O
HO
Adenine
N
NH2
O
N
O
CH2
O
OH
N
H
N
N
Adenine
Arginine
Redundancy in the Code
•
•
Codons code for only 20 words, or amino acids.
The fact that many amino acids are coded for by several
codons is called degeneracy
The Genetic Code
Met-tRNA
Methionine
16 Pu
17
9
A
17:1
13 12 Py 10
1
2
3
4
5
6
U* 7
A
C
C
73
72
71
70
69
68
67
Py 59A*
66
65 64 63 62 C
49 50 51 52 G T C
y
Py
G*
22 23 Pu 25
G
26
2020:120:2A
27
1
28
29
30
31
Py*
Anticodon
Pu
47:16
47:15
43 44
42 45
41 46
47
40
47:1
39
38
Pu*
U
34
U 35
C
A 36
Translation - Initiation
fMet
Large
subunit
E
P
A
UAC
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
Small mRNA
subunit
3’
Translation - Elongation
Polypeptide
Arg
Met
Phe
Leu
Ser
Aminoacyl tRNA
Gly
Ribosome
E
P
A
CCA
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
mRNA
3’
‫ בתא‬RNA ‫סוגי‬
80%
rRNA
10%
tRNA
5%
mRNA
5% snRNA , gRNA
‫ ריבוזומלי‬RNA .1
‫נשא‬
RNA . 2
‫ שליח‬RNA
.3
‫ אחרים‬.4
Genetic engineer method:
1.Restriction Enzymes
2.PCR
Restriction Enzymes (REs)
are endonucleases which cut ONLY
double-stranded DNA that contain a particular
nucleotide sequence (recognition site) ALWAYS in
the same way
Bacterial enzymes, destroy the foreign DNA
Most REs recognise PALINDROMIC sequences
The sequence on one strand reads the same in the
opposite direction on the complementary strand .
GTAATG is not a palindromic DNA sequence
EcoRI
5' - G A A T T C - 3'
3' - C T T A A G - 5'
Application of REs
Gene cloning
Potential" restriction sites "appear in
almost any gene that can snip it out.
The sequences of some artificial
plasmids include a "linker" that contains
dozens
of
restriction
enzyme
recognition sequences within a very
short segment of DNA.
Restriction Enzyme/s
Gene cloning
REs will produce ends that enable the gene to be spliced into a plasmid
Ligation
PCR
• Inventor: 1983 Kary Mullis
– Nobel prize in chemistry in 1993
needs only slightly DNA molecules to produce a
huge range of copies
PCR needs unleast some information of the
gene order (or from some similar gene) to make
the primer
Tools for PCR
A small amount of DNA
Taq DNA Polymerase (or another thermally stable
DNA polymerase)
Nucleotides
Primers
– Two different kind of
– Usually about 20 nucleotides
Temperature
PCR
100
Melting
94 oC
50
0
T i m e
3’
5’
5’
3’
Temperature
PCR
100
Melting
94 oC
50
0
T i m e
3’
5’
Heat
5’
3’
Temperature
PCR
100
Melting
94 oC
50
0
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
T i m e
3’
5’
5’
5’
5’
3’
Temperature
PCR
100
Melting
94 oC
50
0
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
T i m e
3’
5’
Heat
5’
5’
Heat
5’
5’
3’
30x
Temperature
100
Melting
94 oC
50
0
PCR
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
T i m e
3’
5’
5’
5’
5’
5’
5’
5’
5’
3’
30x
Temperature
PCR
100
Melting
94 oC
50
0
3’
5’
5’
T i m e
5’
5’
5’
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
5’
3’
Fragments of
defined length
5’
5’
5’
5’
5’
5’
30x
DNA Between The Primers Doubles With
Each Thermal Cycle
Number
1
2
0
1
Cycles
4
8
16
32
64
2
3
4
5
6
PCR Program
Initial denaturation 95oC (3-5min)
Prior to the first cycle, the DNA is often denatured for an
extended time to ensure that both the template DNA and the
primers have completely separated and are now single-strand
only. Also certain polymerases are activated at this step (hotstart PCR).
Denaturation 95oC (30-60s)
Annealing (1-2min.)
Elongation 72oC
x20-30cycles
Final extension (10min)
To ensure that any remaining single stranded DNA is completely copied.
Identification of PCR product
Identification of PCR product