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 Phosphate

Pyrimidines NH 2 O N N NH N Guanine N N Adenine N N NH 2 N O N O N Cytosine Uracil (RNA) CH 3 N O N O NH N O N O Thymine (DNA) Purines Two 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 Introduction The Central Dogma of Molecular Biology Cell Polypeptide (protein) Translation Ribosome

3’ 5’ 3’ Transcription And Translation In Prokaryotes Ribosome 5’ mRNA RNA Pol.

DNA Cytoplasm Nucleus Eukaryotic Transcription Export G AAAAAA preRNA Transcription Nuclear pores G AAAAAA RNA Processing 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 4 3 = 64 unique words.

SUGAR-PHOSPHATE BACKBONE B A S E S H P O O HO O O CH 2 NH 2 N NH N N HOH P O O HO O O CH 2 NH 2 N N N N H P O OH HO O O CH 2 NH 2 N N N N O A Codon Guanine 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

Methionine Met-tRNA A C U Anticodon

A E Large subunit P Small subunit Translation - Initiation fMet UAC GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

A E Ribosome P UCU Arg Aminoacyl tRNA Phe Leu Met Ser Gly Polypeptide CCA Translation - Elongation GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

סוגי RNA בתא RNA.1 ריבוזומלי rRNA 80% 2. RNA נשא tRNA 3. RNA שליח mRNA 4. אחרים snRNA, gRNA 10% 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 REs Gene 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 DNA Taq DNA Polymerase (or another thermally stable DNA polymerase) Nucleotides Primers –Two different kind of –Usually about 20 nucleotides

Melting 94 o C Temperature T i m e 5’3’ 5’ PCR

Melting 94 o C Temperature T i m e 3’5’ 3’ Heat PCR

Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature T i m e 3’5’ 3’ 5’ Melting 94 o C PCR

Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature T i m e 30x 3’5’ 3’ Heat 5’ PCR

PCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature T i m e 30x 3’5’ 3’ 5’

Fragments of defined length PCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature T i m e 30x 3’5’ 3’ 5’

DNA Between The Primers Doubles With Each Thermal Cycle 0 Cycles Number

PCR Program Initial denaturation 95 o C (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 95 o C (30-60s) Annealing (1-2min.) Elongation 72 o C x20-30cycles

Identification of PCR product