6.1.3 Manipulating genomes

Question 1

PCR

Answer 1

polymerase chain reaction
artificially amplifies (copies) same copy of DNA
used to make enough DNA to test multiple times (e.g. crimes, genetic profiling)

Question 2

DNA primers

Answer 2

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

Question 3

PCR key steps and temps

Answer 3

denaturation - 95°C
annealing - 68°C
elongation - 72°C

Question 4

PCR method

Answer 4

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

Question 5

why Taq DNA polymerase used

Answer 5

obtained from thermophilic bacterium

stable at high temperatures

Question 6

anneal definition

Answer 6

bind by hydrogen bonding

Question 7

applications of PCR

Answer 7

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)

Question 8

PCR vs DNA replication

Answer 8

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

Question 9

DNA electrophoresis method

Answer 9

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

Question 10

stain used in DNA electrophoresis

Answer 10

ethidium bromide

Question 11

electrophoresis of proteins method

Answer 11

same way as DNA

sodium dodecyl sulfate added so protein have equal negative charge and separated by mass rather than charge

Question 12

protein electrophoresis uses

Answer 12

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

Question 13

DNA probe

Answer 13

short single-stranded length of DNA complementary to a section of DNA being investigated

Question 14

how DNA probes are labelled

Answer 14

radioactive marker: 32P in one of Pi revealed by exposure to photographic film after annealing
fluorescent marker: emits colour on exposure to UV light

Question 15

probes uses in locating DNA sequences

Answer 15

identify specific gene needed for genetic engineering
same gene in variety of different genomes
identify presence or absence of allele for particular disease

Question 16

microarray

Answer 16

number of different probes fixed on surface
any mutated alleles anneal to matching fixed probes
labelled with fluorescent markers

Question 17

DNA profiling

Answer 17

method of identifying individuals by DNA

almost all human DNA is the same so not suitable for comparisons

Question 18

STR stands for

Answer 18

short tandem repeats

Question 19

STRs

Answer 19

loci on genome composed of 2-10 bases repeated 5-50 times in a row

Question 20

STRs in comparing DNA

Answer 20

number of times the STR repeats at each loci varies from person to person

Question 21

probability of people having the same STR

Answer 21

1 in 10 per each loci

Question 22

how to find out number of STRs person has for each location

Answer 22

electrophoresis

longer STRs = larger DNA fragments = move less far in electrophoresis

Question 23

how to create DNA profile

Answer 23

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

Question 24

use of DNA profiling

Answer 24

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

Question 25

DNA sequencing definition

Answer 25

process of working out order of bases on a DNA molecule/gene

Question 26

DNA sequencing method (Sanger)

Answer 26

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

Question 27

ddNTP stands for

Answer 27

dideoxynucleotides triphosphates

Question 28

why polyacrylamide gel used instead of agarose gel in electrophoresis

Answer 28

higher resolving power | can separate DNA strands that differ in length by 1 base pair

Question 29

pyrosequencing reaction cascade system method

Answer 29

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)

Question 30

aims of human genome project

Answer 30

identify all genes | work out order of ~3 billion base pairs in human genome

Question 31

benefits of knowing human DNA profile

Answer 31

``` 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 ```

Question 32

comparing genomes between species

Answer 32

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

Question 33

benefits of comparing genomes between species

Answer 33

``` 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) ```

Question 34

comparing genomes between individuals

Answer 34

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

Question 35

epigenetics definition

Answer 35

study of changes in organisms by modification of gene expression rather than changes of genetic code

Question 36

how to obtain required gene in genetic engineering

Answer 36

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)

Question 37

restriction enzymes origins

Answer 37

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

Question 38

place gene into vector method (genetic engineering)

Answer 38

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

Question 39

DNA ligase function

Answer 39

catalyses condensation reactions to join pentose sugar and phosphate groups of DNA backbone

Question 40

different ways of getting vector into recipient cell

Answer 40

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)

Question 41

direct method of introducing gene into recipient method

Answer 41

small pieces of gold or tungsten covered in DNA shot into cell