Summary Molecular Biology of the Cell (5234MOBC6Y) partial exam 1+2
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Vak
(5234MOBC6Y)
Instelling
Universiteit Van Amsterdam (UvA)
Boek
Molecular Biology of the Cell
Complete summary of the course Molecular Biology of the Cell (5234MOBC6Y) from the 1st year of the master biomedical sciences, UvA, compulsory for all tracks. This summary contains all information needed for partial exam 1 and 2 (Van der Spek, Stam, and Brul), and includes all the material from the...
,DNA Replication & Sequencing & PCR
THE CENTRAL DOGMA
- Going from the gene to function: DNA -> RNA -> protein -> metabolite -> phenotype
- Replication (DNA synthesis): DNA -> DNA
- ~ PCR, sequencing
- Transcription (RNA synthesis): DNA -> RNA
- ~ promoters, regulatory factors, splicing
- Translation (protein synthesis): RNA -> protein (made from amino acids)
- ~ RNA structure and stability, ribosomes, post-translational modi cations (=decoration of
proteins)
DNA SYNTHESIS / REPLICATION
- DNA replication is semi-conservative: every new double-stranded molecule consists of 1 old
and 1 new strand
- The old strand is the template (i.e., example) for the new strand
- 1 parental DNA double helix results in 2 identical daughter DNA double helices
- DNA replication always occurs 5’->3’; the template strand is anti-parallel (3’->5’)
- DNA synthesis is the process of the formation of phosphodiester bonds while hydrolyzing
the matching dNTP (deoxyribonucleoside triphosphate) molecule
- The energy comes from the DNA backbone: as the phosphodiester bond forms between
the 5’ phosphate group of the new nucleotide and the 3’ OH of the last nucleotide, 2
phosphates are removed providing energy for bonding
- DNA polymerase synthesizes DNA from a double-stranded ‘primer’
- DNA polymerase requires a stable primer already bound to the template strand to
synthesize DNA, since it needs a pre-existing 3’ OH to elongate the strand
- The replisome is a molecular machine with essential components for DNA replication:
- Topoisomerase: enzyme that regulates the over-/underwinding of DNA by making a nick
in the DNA, thereby releasing the tension of the coiled DNA helix
- Helicase: breaks hydrogen bonds between DNA strands, thereby separating the strands
using ATP
- Primase: RNA polymerase that synthesizes a short RNA primer that is complementary to
a single-stranded DNA template, which is needed by DNA polymerase
- After elongation of the strand, the primer is removed by a 5’ to 3’ exonuclease and
replaced by DNA
- 2 DNA polymerases: one synthesizes leading strand, while the other synthesizes the
lagging strand
- Leading strand: template (antisense) strand that goes 3’->5’
- Replication occurs continuously 5’->3’
- 1 RNA primer is made at the origin
- Lagging strand: anti-parallel template strand (coding/sense) that goes 5’->3’
- Replication occurs discontinuously 5’->3’
- Many RNA primers are required to synthesize Okazaki fragments (=small 5’->3’
DNA fragments)
- Ligase: covalently links adjacent Okazaki fragments together
MISTAKES IN DNA SYNTHESIS
- Mistakes in DNA synthesis can be restored through ‘proofreading’ activity of the DNA
polymerase complex
- Without proofreading (5’->3’ polymerization): 1 error per 105 nucleotides
- With proofreading: 1 error per 102 nucleotides
- Proofreading: DNA polymerase recognizes a mismatched nucleotide by 3’->5’
proofreading, removes it, replaces it with the correct nucleotide, and allows synthesis
to continue 5’->3’
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, - With strand-directed mismatch repair: 1 error per 103 nucleotides
- Strand-directed mismatch repair: mismatch proofreading proteins (MutS and MutL)
recognize and bind to an error in newly-made strand -> DNA scanning detects a nick
in new DNA strand -> strand removal containing the error -> repair DNA synthesis
using template
- Occurs after DNA replication
- These 3 mechanisms combined give 1 error per 1010 nucleotides
- If the error would be 0, there would be no evolution: allowing some errors is a good
thing
DNA MUTATIONS
- Chemical changes in DNA bases can cause mutation in the DNA
- Depurination: involves the removal of a purine (G/A) from the DNA, leaving only the sugar
phosphate of the nucleotide
- Results in 1 mutated daughter helix with an A-T/G-C deletion, and 1 unchanged daughter
helix
- Is the most common DNA mutation
- Deamination: involves the removal of an amino group
- Results in 1 mutated daughter helix with a change in base pair, and 1 unchanged daughter
helix
- C -> T
- Deamination of C results in a U -> the mutated daughter strand will have a G to
A conversion in the new strand while U will be repaired into a T -> C to T
conversion at the position of the deaminated C
- A -> G
- G stops replication (stutter/deletion)
DNA SEQUENCING
- DNA sequencing (whole genome) is used to determine the order of the bases A, C, G, and T
- Sanger sequencing / dideoxy sequencing: DNA synthesis while incorporating chain
terminators in separate reaction for the bases A, C, G, and T
- A chain terminator (dideoxynucleotide) lacks the 3’ OH necessary for strand elongation,
thereby preventing extension of the DNA strand, resulting in DNA fragments
- With a high concentration of all dNTPs and low concentrations of ddATP, ddCTP, ddGTP
and ddTTP, in separate reactions DNA synthesis will continue, but occasionally synthesis
will stop when a dideoxynucleotide is incorporated
- The oligonucleotide primer (needed by DNA polymerase) determines the start of the
reaction
- With 4 separate reactions (ddATP/ddCTP/ddGTP/ddTTP + DNA polymerase) and a
labelled (radioactive or uorescent) primer, fragments of di erent lengths are generated:
these fragments are separated alongside using electrophoresis, and every visible fragment
represents a termination in the DNA synthesis
- The DNA sequence is read from the bottom of the gel upward
- E ciency of manual sequencing methods:
- Radioactive nucleotides in 4 lanes: ~8000 bases per day
- Fluorescent nucleotides in 4 lanes: ~10,000 bases per day
- Fluorescent nucleotides in 1 lane: ~30,000 bases per day
- Nucleotides in capillary gel (1-96 wells): ~150,000 bases per day
- Genome sequencing / hierarchical shotgun sequencing: multiple copies of the genome are
randomly fragmented -> the nucleotide sequence of each clone is stitched together, using the
overlaps between clones as a guide
- Works well for small genomes that lack repetitive DNA
- Uses contigs: assembly of smaller DNA sequences into 1 continuous strand
- Needs markers such as restriction enzymes
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