theme 4: modules 3 and 4 Flashcards
DNA sequencing is important..
to understand a genes function & to figure out the amino acid sequence & the function of its product.
genome sequencing phases
1: sequence DNA
2: assemble the sequences
3: annotate the sequences
Dideoxynucleotides
lack an OH group on the 3’ carbon of the sugar, therefore DNA polymerase cannot add another nucleotide to the growing DNA strand once a ddNTP has been added.
Replication Stops at -
chain terminator
sequence of the template strand is unknown, elongation of daughter strand stops wherever a dideoxynucleotide terminator is incorporated at the 3’ end
Each Terminator is
Fluorescently labelled - which reveals the identities of the successive dideoxyribonucleotides that terminate strand elongation, allows template sequence to be deduced
Assembly phases
Phase 1: Sequenced DNA Fragments (in pieces)
Phase 2: Overlapping DNA Sequences (same ones are overlapped)
- like sentence fragments in english, can be assembled in the correct order according to their overlaps and the original complete sentence reconstructed
Phase 3: Annotation
-any double-stranded region of DNA can be read in 6 ways - 3 reading frames using each of the two single strands as a template
- 6 reading frames for any double stranded DNA sequence, but only 1 frame codes for the protein
-Sequences that might form typical RNA secondary structures (regions of tRN, form hairpin structures in which the molecule folds back on itself)
- identifying the locations of genes & all of the coding regions in a genome & determining what those genes do.
Causes and Consequences of DNA damage
Causes: X-rays, Oxygen radicals
UV light, X-rays, Replication
errors, base-excision repair (BER),
Nucleotide- excision repair (NER),
Mismatch repair
Consequences: Cell death, Cancer, Aging, Disease
Mismatch repair
- identify mistake
- a repair enzyme breaks the backbone some distance away
-another enzyme removes successive nucleotides, including the one w/the mismatched base
-other enzymes close the gap by new DNA synthesis, using the intact DNA strand as a template
Base excision repair
-uracil in DNA signals repair process
-DNA uracil glycosylase cleaves the uracil from the deoxyribose sugar (apurinic/apyrimidinic AP endonuclease)
- AP endonuclease cleaves the backbone and removes the sugar
-other enzymes close the gap by new DNA synthesis, using intact nucleotide opposite the site, as a template
Nucleotide excision repair
- one or more damaged bases signals the repair process
- an enzyme cleaves the DNA backbone at sites flanking the damage
-region w/ damaged bases is removed - gap filled by new DNA synthesis, using up gapped strand as template
Point mutation
-when a G is incorporated into the daughter strand opposite the T and is not corrected by the proofreading function
-In the next round of replication, the G specifies a C in the opposite strand. The new C-G pair is replicated as faithful as the original T-A pair and the mutation is now present in this cell lineage
3 effects of point mutations:
a) non mutant (normal B-globin)
b) synonymous/silent mutation (nucleotide substitution that does not change the amino acid)
c) nonsynonymous/missense mutation (nucleotide substitution that changes the amino acid is a nonsynonymous mutation)
Why would Silent Mutations not cause a change in amino acid sequence?
Due to the redundancy in the genetic code, a single nucleotide change in the 3rd position of the codon may not change the amino acid for which it is coded.
If the amino acid sequence does not change…
…then the phenotype does not change
Nonsense mutation
nucleotide substitution that creates a stop codon
