D1.1-1.3

pcr

production of large quantities of dna

1. denaturation : break down the hydrogen bond (75 degree)

2. annealing : dna primer attach to the 3’ end of target sequence

3. elongation : tag polymerase attaches to dna primer and copies the strand

gel electrophoresis

1. dna fragments are loaded into the wells in one side of the porous gel

2. negative electric current is applied

3. since dna are slightly negative due to their phosphate group, it migrates to the other end

4. smaller fragments move faster and further while larger fragments move slower

5. result in a separation of dna fragments, creating a dna profile

dna replication

1. Helicase separates the strands by breaking the hydrogen bonds. Each strand acts as a template strand

2. RNA primase adds RNA primer complementary to the template strand

3. DNA polymerase III adds dNTP to the 3’ end of primer according to complementary base pairing (adenine-thymine, guanine-cytosine)

4. Since DNA synthesis must be in 5’ the 3’ direction, the leading strand is continuous, while the lagging strand is discontinuous. Okazaki fragments are formed.

5. DNA polymerase 1 rephases rna primer with DNA

6. Dna ligase covalently bonds Okazaki fragments

transcription:

dna - mrna

1. rna polymerase binds with DNA and breaks hydrogen bond

2. antisense strand acts as the template

3. rna polymerase adds rna nucleotide according to complementary base pairing

4. continue until reached terminator sequence, mrna strand detaches from DNA

enters post-transcriptional modification

post transcriptional modification

• splicing of intron (non-coding dna)

• alternative splicing ( different mRNA- code for different proteins - diverse antibodies - enhance immune system and protein diversity)

• add methyl group to the 5’ head of mRNA

• identification of the ribosome

• protection from exonuclease

• add poly A tail to the 3’end of mRNA

• provide mobility (nucleus - cytoplasm)

translation

1. mRNA binds to the small ribosome subunit

2. first tRNA with anticodon complementary to the start codon(AUG) binds to the p site

3. large ribosomal subunit binds , create functional ribosome

4. second tRNA with anticodon complementary to the second codon binds to the A site

5. first amino acid in tRNA forms peptide bond with the second amino acid. Attaches to second amino acid, detach from first tRNA

6. ribosome moves from codon to codon , first tRNA enters E site, leaves the ribosome . second tRNA moves to P site and third tRNA comes into A site

7. this continues until reaches STOP codon, release factor binds to A site and polypeptide is release from tRNA

8. ribosome dissociates, reused for 2nd translation

base substitution mutation

sickle cell anemia

• DNAchanges from GAG → GTG

• tRNA adds the wrong amino acid to the poly peptide chain GLU → VAL

• result in a change in protein

• RBC being distorted - crescent shape, lowers the ability to transport oxygen

post transcirptional modification example

insulin

pre proinsulin

• enters endoplasmatic recticulm , signal sequence is removed

Proinsulin

• disulfide bridge formed

• moves into golgi apparatus, c peptide removed

Insulin

proteasomes

break down unused protein to amino acids to make new proteins

base substitution mutation (3)

1. nonsense - change in dna sequence - premature stop codon

2. missense - change in amino acid - sickle cell anemia

3. silent - degeneracy : codon codes for same amino acid

deamination example

cytosine mutates to uracil

factor that affects mutation

1. radiaton → breaks hybrogen bond, base mutates

2. chemical mutagens

3. exposure to environment agents( mutagens)

gene knockout

investigate the function of the gene

crispr cas 9

gene editing system

• alter dna by deletion, substitution and insertion of bases

crispr cas 9 components

1. cas 9 : restriction enzyme that cuts dna at specific sequence

2. dna sequence

3. guide rna:

   • guides cas9 to dna sequence

   • complementary to dna sequence

Crispr cas 9 process

1. Cas 9 bound to gRNA(with sequence complementary to target sequence)

2. gRNA guides Cas 9 to the target sequence

3. Cas 9 cuts DNA in the target location

4. knock out a gene → no longer makes protein / insert DNA

example of crispr cas 9

sicilian rouge High GABA tomatoes

• reduce ripening, reduce blood pressure, promotes relaxation

genetic research

epigenetics

change in gene expresson without altering dna sequence

epigenetic tags

chemical tags on dna and histone to promote or inhibit the binding of rna polymerase to promoter

1. methylation: tightly packed, dna polymerase cannot bind to promoter , no gene expresson

2. acetylation: loosely packed, dna polymerase binds to promoter, enable gene expression

epigenetic inheritance

removal of epigenetic tags to promote differentiaton

some retain ( imprinted genes)

imprinted genes example

tigon

liger → (male lion x female tiger ) larger size imprinted

lac opeon

→ no lactose : lac repressor binds to the operator. RNA polymerase cannot transcribe

→ yes lactose : lactose binds to repressor, detaches it from the operator, and allows RNA polymerase to bind to promoter , transcripton occurs