BIOL1130 Lecture Notes - Lecture 14: Taq Polymerase, Electric Field, Mitochondrion
28 May 2018
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8.1.1 PCR
• Denaturation -Genomic DNA ( templates ) are heated to 95°C to denature the templates
( break H bonds)
• Annealing - Mixture is cooled so that primers can hybridize to the complementary sequences
ut larger DNA fragets dot rejoi
• Elongation - Taq DNA polymerase replicate each strand
• After about 30 repeats, the repeated fragments can be cloned
• How does it start? Primers attach to template strand at low temp, to hybridise
complementary sequences
8.1.2 DNA fingerprinting
• primers
DNA Sequences – e.g. AGCTAGCAT
• allows evolutionary relationship comparisons
• application – DNA dissociation, DNA rehybridisation
DNA replication in the nucleus (eukaryotes) – link week 9 chap 10
Requirements-
1. DNA template
2. 3 –OH group (on a primer)
• because primers attah to the 3 –OH group to determine which part of the genome is to
be replicated
3. Enzyme/Protein Catalyst –
• DNA polymerase, fx to extend primers and add nucleotides, DNA polymerase builds new
strands
4. dNTP precursors-building blocks, dATP,dCTP,dGTP,dTTP
5. DNA ligase glues at sticky ends
• DNA replication overall process- 4 requirements, put buffer, magnesium, put to PCR
machine.
• DNA replication occurs in test tube, depends on suitable °C and salinity.
• Therefore DNA replication works with PCR
• Function of primers- attach to –OH at 3 , deterie hih part of geoe is to e
amplified
DNA replication application
• Small amount of DNA is amplified from PCR, now, DNA fingerprinting can be used to profile
idiiduals DNA.
• In DNA fingerprinting, fragments that are amplified previously are separated by gel
electrophoresis
o DNA is put into the gel with some buffers
o Electric field is applied, fragments move from – to + end, shorter ones move further
and faster.
• The difference in profiles show polymorphism, can be used to compare.
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Primers attach to template strand at low temp, to hybridise complementary sequences. Dna sequences e. g. agctagcat: allows evolutionary relationship comparisons, application dna dissociation, dna rehybridisation. Dna replication in the nucleus (eukaryotes) link week 9 chap 10. Dna replication application: small amount of dna is amplified from pcr, now, dna fingerprinting can be used to profile i(cid:374)di(cid:448)idual(cid:859)s dna. Molecular phylogeny- dna melting curves: dna melts means it becomes single stranded ( dissociated ) Origin of mitochondria: origin- rickettsia , genome is most related to mt genes, rickettsia (origin of mt) has smaller genome than any bacteria, genomes of organelles are small if compared to modern cyanobacteria/bacteria. Origin of plastid ( chloroplast, pigments, thylakoid membranes: close relative of cyanobacteria, distantly related to mitochondria, chloroplasts and mitochondria have their own genes, and were independently derived from different ancestral prokaryotes. Chloroplasts: have multiple circular dna like mitochondria. Share features with prokaryotes, this distinguish them from eukaryotes. Their ri(cid:271)oso(cid:373)es rese(cid:373)(cid:271)le prokaryotes(cid:859) ri(cid:271)oso(cid:373)es (cid:894) s(cid:373)all 70s(cid:895)
