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
DNA profiling
method of identifying individuals by DNA
almost all human DNA is the same so not suitable for comparisons
STR stands for
short tandem repeats
STRs
loci on genome composed of 2-10 bases repeated 5-50 times in a row
STRs in comparing DNA
number of times the STR repeats at each loci varies from person to person
probability of people having the same STR
1 in 10 per each loci
how to find out number of STRs person has for each location
electrophoresis
longer STRs = larger DNA fragments = move less far in electrophoresis
how to create DNA profile
obtain DNA from all people to be compared e.g. from saliva/hair
amplify DNA using PCR
DNA from all people cut into different size fragments using same restriction enzymes
DNA from different people will be different sizes because the number of repeats of STRs will vary
DNA fragments separated based on size using electrophoresis
people to be compared are loaded into different wells
banding pattern examined (smaller fragments move further) and compared
use of DNA profiling
maternity/paternity testing (half of STRs on DNA profile come from mother and father)
studying evolutionary relationships (more similar banding pattern = more closely related)
crime scene investigations
DNA sequencing definition
process of working out order of bases on a DNA molecule/gene
DNA sequencing method (Sanger)
DNA is amplified (PCR)
DNA heated to denature to form template strand and complementary strand
primer anneals to template strand
primed DNA dispersed equally to 4 test tubes containing DNA polymerase and all DNA nucleotides added to test tubes
one different type of ddNTP is added to each test tube
polymerase attaches nucleotides to template strand until ddNTP is base paired, sequence is terminated as ddNTP lacks -OH group at 3’ carbon
DNA fragments of different lengths formed across all reaction vessels
run each test tube’s contents separately in electrophoresis
read the last letter of each strand from shortest to longest to get complementary base sequence
ddNTP stands for
dideoxynucleotides triphosphates
why polyacrylamide gel used instead of agarose gel in electrophoresis
higher resolving power
can separate DNA strands that differ in length by 1 base pair
pyrosequencing reaction cascade system method
DNA polymerase adds bases to DNA, forming pyrophosphate
ATP sulfurylase converts pyrophosphate into ATP
luciferin is oxidised by luciferase (requires ATP) and generates a light signal, recorded as pyrogram peak
apyrase removes any unincorporated nucleotides remaining in reaction
pyrogram peak proportional to number of nucleotides added by polymerase (more pyrophosphate = more ATP = more light)
aims of human genome project
identify all genes
work out order of ~3 billion base pairs in human genome
benefits of knowing human DNA profile
improved genetic testing location of genes that may be linked to increase chances of inheriting a disease new gene therapy humans’ evolutionary relationships personalised medicines synthetic biology
comparing genomes between species
evolutionary relationships explored by comparing similarities/differences in genomes
beneficial/important genes conserved by evolution (common in most/all organisms)
idenfity altered genes that give rise to differences between organisms
benefits of comparing genomes between species
medical research (compare genome of pathogenic vs non-pathogenic bacteria to identify genes responsible for disease) identification of evolutionary relationships between species (identifying altered genes and similar genes)
comparing genomes between individuals
methylation of DNA influences gene regulation, mapping methylation helps understand how diseases develop in similar individuals
investigate relationship between which genotypes cause which phenotypes
mapping early human migration by comparing genomes around the world
medical advances made by possibly producing drugs specific to an indiviuals genome to maximise its effect
epigenetics definition
study of changes in organisms by modification of gene expression rather than changes of genetic code
how to obtain required gene in genetic engineering
mRNA removed from cells expressing gene
used as template for reverse transcriptase to form complementary DNA strand
primers and DNA polymerase turn this into a double stranded length of DNA coding for original protein
automated polynucleotide synthesiser builds fene (if nucleotide sequence known)
PCR primers used to amplify gene (if sequence known)
DNA probe used to locate gene (cut out using restriction enzymes)
restriction enzymes origins
from bacteria and archae to protect from phage virus attacks
cut up foreign viral DNA
their own DNA is protected as it is methylated at recognition site
can be blunt or sticky
Mg2+ ions may be needed as cofactor
place gene into vector method (genetic engineering)
cut open plasmid using restriction enzymes (the same ones used to isolate the gene with)
leaves complementary sticky ends
sticky ends of genes and plasmid anneal and hydrogen bonds form between complementary bases
DNA ligase function
catalyses condensation reactions to join pentose sugar and phosphate groups of DNA backbone
different ways of getting vector into recipient cell
heat shock treatment (bacteria alternated between 0-42°C with CaCl, membranes becomes more porous)
electroportation (high voltage pulse applied to cell to disrupt membrane)
electrofusion (electrical fields help introduce DNA into cells)
transfection (DNA packaged into bacteriaphage, which can transfect whole cell)
recombinant plasmids (plasmids inserted back into bacteria that infect plant cells)
direct method of introducing gene into recipient method
small pieces of gold or tungsten covered in DNA shot into cell
