molecular genetics Flashcards
1
Q
properties of DNA
A
- store complex information
- replicate faithfully
- relatively stable
- mutate occasionally to produce variation
- be translated into proteins to produce phenotype
2
Q
PCR
A
- polymerase chain reaction
- en vitro amplification of specific DNA/RNA fragments
- used in DNA profiling, diagnostics, analysis of ancient DNA and genetic engineering
- cycle repeats 20-40 times, producing millions of copies
3
Q
PCR setup
A
- synthetic primer = short strands of complementary RNA (oglionucleotide) specific to sample. Anneals to unwound DNA to indicate to Taq polymerase where to start, the DNA that is amplified is the sequence between primers
- sample
- Taq polymerase
- free nucleotides (dNTPs)
- sterile water
4
Q
process of PCR
A
- denaturation, DNA heated to ~95C, breaking H bonds
- annealing, temperature drops to ~55C, primer bonds to DNA section to be replicated
- extension, 70-75C, Taq polymerase binds at primer, free nucleotides align, Taq creates phosphodiester bonds
5
Q
structure of DNA
A
- nucleotide subunits containing phosphate, deoxyribose and base A,T,C,G
- antiparallel strands, molecule has 5’ end and 3’ end
6
Q
nucleotide base pairs
A
- A and T bind with 2 H bonds
- C and G bind with 3 H bonds, so are more stable
- a higher proportion of CG base pairs in DNA means that more energy is required to split it
7
Q
semi-conservative DNA replication
A
- DNA helicase breaks H bonds between complementary base pairs, topoisomerase unwinds helix
- both strands used as template, complementary base pairing occurs
- DNA polymerase forms phosphodiester bonds between adjacent nucleotides (condensation reaction)
8
Q
replication bubbles
A
- DNA synthesis happens at multiple places on the chromosome at once in replication bubbles
- bubbles merge as replication forks meet each other
9
Q
discontinuous replication
A
- DNA polymerase can only add nucleotides to 3’ end
- strands are antiparallel
- leading strand continuously replicated (direction or DNA synthesis same as replication)
- lagging strand discontinuously replicated, direction of DNA synthesis is opposite to replication
- produces Okazaki fragments 100-200 nucleotides long, DNA ligase seals gaps between fragments after synthesis
10
Q
primers
A
- short strands of single stranded complementary RNA produced by enzyme primase
- anneal to unwound DNA to initiate synthesis, as DNA polymerase can only add nucleotides to an existing strand
- removed after synthesis and replaced by extension of adjacent replicated strand
11
Q
telomeres
A
- at chromosome ends there is a gap where primer is removed, so the chromosome is shortened every time it is replicated
- telomeres are a cap on the end of chromosomes made up of repeated DNA sequences that don’t code for anything, so can be lost in replication
12
Q
telomerase
A
- enzyme that extends telomeres
- only active in early embryonic cells
13
Q
Hafflick limit
A
amount of divisions adult somatic cells can undergo before DNA that code for proteins are lost
14
Q
double strand breaks
A
- can be caused by radiation or chemical mutagens
- occurs in prophase I of meiosis
- homologous chromosome used as template for repair
15
Q
homologous recombination
A
- homologous chromosomes line up during prophase I
- enzyme (Spo11) creates double strand break in one of the chromosomes
- nuclease enzyme (MRX) digests sections either side of breaks, creating 3’ overhang
- strand invasion (enzymes Dmc1 and Rad51), free 3’ end invades intact chromosome
- DNA polymerase adds nucleotides back on using homologous chromosome as a template for repair
- 2 Holliday junctions formed between DNA molecules, direction that Holliday junctions are cut determine whether recombination occurs
