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From DNATo RNA
To Protein
OH
OCH2
Sugar
HOH
A Nucleotide
NH2
N
N N
N
BaseP
O
OH
HO O
Phosphate
Pyrimidines
NH2
O
N
N NH
N
Guanine
N
N
Adenine
N
N
NH2
N O
NH2
N O
NH2
NCytosine
Uracil(RNA)CH3
N ON
O
NH
N ON
O
NH
Thymine(DNA)
PurinesTwo Families of Bases
2 H- bonds for A:T 3 H-bonds for G:C
Hydrogen bonds
Ribose Deoxyribose
Genomes vary in size
DNA
mRNA
Transcription
IntroductionThe Central Dogma of Molecular Biology
Cell
Polypeptide(protein)
TranslationRibosome
3’
5’
5’
3’
Transcription And Translation In Prokaryotes
Ribosome
5’
mRNA
RNAPol.
DNA
Cytoplasm
Nucleus
Eukaryotic Transcription
ExportG AAAAAA
preRNA
Transcription
Nuclear pores
G AAAAAA
RNAProcessing
mRNA
Translation
Ribosome
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.
SU
GA
R-P
HO
SP
HA
TE
BA
CK
BO
NE
B A
S E
S
H
PO
O
HO
O
O
CH2NH2N
NH
N
N
HOH
P
O
O
HO
O
O
CH2
NH2
N
N
N
N
H
P
O
OH
HO
O
O
CH2
NH2
N
N
N
N
O
A Codon
GuanineGuanine
AdenineAdenine
AdenineAdenine
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
Methionine
Met-tRNA
U*
9
262223Pu
16
12Py 10
25
20:1
G*
17:1
Pu
A20:2
1713
20G
A5051
656463
G
62
52
CPu
59
A*
C
Py
T49
39
4142
31
2928
Pu*
43127
U35
38
36
Py*
34
403047:1
47:15
46
Py47:16
4544
47
73CCA
707172
66676869
321
7654
A CU
Anticodon
AE
Large subunit
P
Small subunit
Translation - Initiation
fMet
UACGAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA5’mRNA
3’
AE
Ribosome P UCU
Arg
Aminoacyl tRNA
PheLeu
Met
SerGly
Polypeptide
CCA
Translation - Elongation
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA5’mRNA
3’
בתא RNAסוגי
RNA .1 ריבוזומלי rRNA
80%
2 . RNA נשאtRNA
3 .RNA שליחmRNA
snRNA , gRNA. אחרים 4
10%
5%
5%
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
EcoRI
5' - G A A T T C - 3' 3' - C T T A A G - 5'
The sequence on one strand reads the same in the opposite direction on the complementary
strand. GTAATG is not a palindromic DNA sequence
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.
Application of REsGene cloning
Restriction Enzyme/s
Gene cloning
REs will produce ends that enable the gene to be spliced into a plasmid
Ligation
• 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
PCR
Tools for PCR
A small amount of DNATaq DNA Polymerase (or another thermally
stable DNA polymerase)NucleotidesPrimers
– Two different kind of– Usually about 20 nucleotides
Melting
94 oC
Tem
pera
ture
100
0
50
T i m e
5’3’
3’5’
PCR
Melting
94 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’
Heat
PCR
Melting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’5’
5’
Melting94 oC
PCR
Melting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’
PCR
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’5’
5’
5’
5’
5’
5’
Fragments of defined length
PCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
5’
5’
5’
5’
DNA Between The Primers Doubles With Each Thermal Cycle
0Cycles
Number1
3
8
2
4
1
2
4
16
5
32
6
64
PCR ProgramInitial 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 (hot-start PCR).
Final extension (10min) To ensure that any remaining single stranded DNA is completely copied.
Denaturation 95oC (30-60s)Annealing (1-2min.)Elongation 72oC
x20-30cycles
Identification of PCR product
Identification of PCR product