from dna to rna to protein. oh o ch 2 sugar h oh a nucleotide nh 2 n n n n base p o oh ho o...

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