vector cloning vs PCR

vector cloning
-slower than PCR
-requires restriction enzymes, ligase, and vector DNA
-uses plasmid
-injection of plasmid into bacterial cell
-proteins can be produced

PCR
-faster than vector cloning
-used to amplify a target sequence
-only requires a single copy of DNA
-can be done with a machine
-forensic uses
-If the first copy is contaminated, the rest would also be contaminated.

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What is a gene?
Any region of DNA that codes for a specific amino acid sequence-->forms RNA/polypeptides.

...


What is a fish?
beeeeeeep.

Mutations

A. Substitutions
1. Silent mutation
-no effect on overall structure
-e.g. UUU and UUC both code for Phe
2. Missense mutation
-code for a different type of amino acid
e.g. UUU-->UUG       Phe-->Leu
3. Nonsense mutation
-amino acid to non-functioning protein/early stop
-e.g. UAC-->UAG     Tyr-->Stop

Example:
Wildtype: AUG CUU UGC CCU UAA
                  M       L      C       P    stop
Missense: AUG CUU UGG CCU UAA
                  M       L       T      P
Nonsense: AUG CUU UGA CCU UAA
                   M      L     stop

B. Frameshifts
4. Insertion
-addition of nucleotide(s)
5. Deletion
-loss of nucleotide(s)

Example:
Wildtype: THE FAT CAT ATE THE RAT
Insertion: THE FAR TCA TAT ETH ERA T
Deletion: THE FAC ATA TET HER AT

Translation :S

Translation: Ribosomes use mRNA as blueprint to synthesize amino acids which form proteins.

-64 codon combinations
-61 code for amino acids
-3 code for stop codon
-20 amino acids
-Wobble Effect: 3rd position of codon is flexible
-->prevents mutations

1. Initiation
i. Ribosome recognizes the 5' cap and binds to it. It contains a large and a small subunit.
ii. Ribosome moves along mRNA.
iii. Translation begins when ribosome reads AUG (start codon).
iv. tRNA delivers Met to the P-site. There is an anti-codon on the tRNA, which is complementary to the codon on mRNA.

2. Elongation
i. A 2nd tRNA carrying an amino acid enters the A-site. Hydrogen bond is formed between the codon and the anicodon (by elongation factors).
ii. Peptide bond is formed between the amino acids.
iii. The petide chain is transfered to the A-site. 1st tRNA leaves the P-site.
iv. tRNA moves from A-site to P-site with the codon (translocation). New codon is available at the A site. 1st tRNA leaves the ribosome at the E-site.
Note: mRNA is read from 5' to 3', codon by codon.

3. Termination
i. Translation stops when the ribosome reads the stop codon (UGA, UAG, UAA).
ii. Release factor hydrolyzes the bond between the polypeptide and the tRNA at the P site.
iii. The polypetide is released :)

Transcription :S

Transcription: Copying of genetic information from DNA to mRNA

1. Initiation:
TATA box, transcriptioin factors (TFs), and RNA polymerase II form the initiation complex upstream of the DNA molecule.
Upstream --> Promoter (sequence of DNA)
i. TATA box is present and is recognized by transcription factors (proteins) in eukaryotes.
ii. RNA polymerase binds to the transcription factors.
iii. Initiation complex is formed.
iv. Initiation complex signals transcription.

2. Elongation
i. RNA polymerase binds to the promoter and unwinds the DNA double helix.
ii. DNA template is read from 3' to 5', and pre-mRNA strand elongates from 5' to 3'.
 
coding strand=sense=non-template
-not used for transcription
-similar to pre-mRNA, except pre-mRNA has U instead of T
non-coding strand=antisense=template
-complementary to pre-mRNA
-read from 3' to 5'
Note: TATA box is not transcribed.

3. Termination
i. Transcription stops at AAUAAA

4. Modifications
i. 5' cap
-protects mRNA from hydrolytic enzymes
-"attach here" signal for ribosomes
ii. poly-A tail at the 3' end
-inhibite hydrolysis
-facilitate ribosome attachment
-facilitate export of mRNA
iii. RNA splicing
introns=non-coding regions
extrons=code for amino acids
-pre-mRNA+snRNP+proteins-->spliceosomes
-snRNA acts as rybozyme and pairs with nucleotides at the ends of introns
-introns form loops and are excised
-exons are spliced together