6.1.3 Manipulating genomes Flashcards
PCR
polymerase chain reaction
artificially amplifies (copies) same copy of DNA
used to make enough DNA to test multiple times (e.g. crimes, genetic profiling)
DNA primers
10-20 bases of single stranded DNA
binds 2 sections of DNA so DNA polymerase can bind
DNA polymerase can’t bind to single strands
PCR key steps and temps
denaturation - 95°C
annealing - 68°C
elongation - 72°C
PCR method
DNA sample mixed with nucleotides, primers, Mg2+ and Taq DNA polymerase
denaturation:
mixture heated to 95°C
breaks hydrogen bonds between complementary base pairs
forms 2 single DNA strands
annealing:
mixture cooled to around
cooled to 68°C
primers anneal to one end of each single strand of DNA
forms small section of double-stranded DNA at end of each single-stranded DNA
elongation:
temperature raised to 72°C
Taq DNA polymerase binds to double-stranded section of DNA
catalyses addition of DNA nucleotides to single strand DNA (builds from 5’ to 3’)
forms a new double strand of DNA
repeat
why Taq DNA polymerase used
obtained from thermophilic bacterium
stable at high temperatures
anneal definition
bind by hydrogen bonding
applications of PCR
tissue typing (donor and recipient tissues be typed prior to transplant, reduces risk of rejection)
detecting oncogenes (if mutation for cancer found, medication tailored to patient)
detecting mutations (look for mutations in genome that cause genetic diseases (can be done in parents/embryos))
identifying viral infections (detect viral genome amongst host cells’ DNA)
forensic science (small quantities of DNA amplified for DNA profiling)
research (analyse DNA from extinct ancient sources)
PCR vs DNA replication
differences:
temperature vs helicase to separate complementary strands
DNA primers needed vs not needed for polymerase to join and replication to begin
doesn’t vs does copy whole chromosome
repeats immediately after one cycle vs every cell cycle
Taq DNA polymerase at 95°C temperatures vs DNA polymerase at body temperature
artificial vs natural
similarities:
both require polymerase, both replicate DNA
DNA electrophoresis method
small amounts of DNA amplified using PCR
DNA cut into smaller fragments using restriction enzymes (same restriction enzymes used to cut fragments from any individuals involved in identification for forensics)
fragments placed into wells at end of gel plate where cathode will be
plate immersed into tank filled with buffer solution and electric current passed through tank (1-2 hours)
DNA is negatively charged (due to phosphoryl groups), attaches to anode on other end of plate, molecules diffuse along gel to other end
shorter fragments move further in same period of time than longer ones
banding pattern invisible so DNA stained with ethidium bromide and viewed under UV light
stain used in DNA electrophoresis
ethidium bromide
electrophoresis of proteins method
same way as DNA
sodium dodecyl sulfate added so protein have equal negative charge and separated by mass rather than charge
protein electrophoresis uses
analyse proteins by mass in blood to diagnose medical conditions e.g.
sickle cell anemia
diseases where fetal haemoglobin is higher than they should be
DNA probe
short single-stranded length of DNA complementary to a section of DNA being investigated
how DNA probes are labelled
radioactive marker: 32P in one of Pi revealed by exposure to photographic film after annealing
fluorescent marker: emits colour on exposure to UV light
probes uses in locating DNA sequences
identify specific gene needed for genetic engineering
same gene in variety of different genomes
identify presence or absence of allele for particular disease
microarray
number of different probes fixed on surface
any mutated alleles anneal to matching fixed probes
labelled with fluorescent markers