6.1.3 manipulating genomes

Question 1

what are the components needed for Sanger sequencing
6.13(a)

Answer 1

· The template DNA whose sequence is being investigated

· DNA polymerase

· Nucleotides A T C and G

· A primer, a short single stranded RNA molecule that binds to the template DNA and allows DNA polymerase to bind

· Special “chain-terminating” versions of A, T, C and G, each labelled with a different colour of fluorescent dye

Question 2

What is DNA sequencing
6.1.3(a)

Answer 2

DNA sequencing is the process of finding out the nucleotide base sequence of a section of DNA.

Question 3

describe the steps of Sanger sequencing
6.1.3(a)

Answer 3

1. Combine all of the components above in a tube.
2. Heat the tube so that the template DNA separates into two strands.
3. Cool so that the primer can anneal (complementary base pair) to the template DNA
4. Heat again so that the free nucleotides A T C and G bind to the template DNA
5. DNA polymerase forms phosphodiester bonds between the free nucleotides
6. If a chain-terminating nucleotide was added, DNA polymerase attaches it to the growing chain, and then falls off so the chain is terminated prematurely, forming a fragment
7. Repeat for many, many, many cycles so that every possible fragment is made

Question 4

how are the fragments then separated
6.1.3(a)

Answer 4

gel electrophoresis
smallest fragments move through gel fastest

Question 5

why does sanger sequencing take a long time
6.1.3(a)

Answer 5

Sanger sequencing takes a long time because many, many cycles must be run before every possible fragment is made

Question 6

how does high-throughput sequencing work
6.1.3(a)

Answer 6

High-throughput sequencing involves first digesting the template DNA into lots of small fragments. All of the fragments are sequenced at the same time.

Question 7

Benefits of high throughput sequencing over Sanger sequencing

Answer 7

automated very rapid and cheaper

Question 8

how does nanopore sequencing work
6.1.3(a)

Answer 8

The template DNA passes through a tiny protein channel (the nanopore). As it moves through, microelectrodes measure the change in electrical current around the pore. The current changes in a predictable way depending on whether A, T, C or G is passing through

Question 9

what are the 3 benefits of DNA sequencing
6.1.3(b)

Answer 9

1. genome wide comparisons between individuals and species to reveal their relatedness
2. Prediction of amino acid sequences of genes to reveal tertiary structure of polypeptides
3. useful for synthetic biology to develop new drugs

Question 10

what are single nucleotide polymorphisms (SNPs)
6.1.3(b)

Answer 10

places on DNA where substitution mutations occur these can be silent, beneficial oe delerious

Question 11

what is bioinformatics and computational technology
6.1.3(b)

Answer 11

storage and analysis of large amounts of information about DNA and RNA sequences AND protein amino acid sequences using computer software.
this allows you to develop algorithms.

Question 12

what is synthetic biology
6.1.3(b)

Answer 12

using information about base sequences to alter the genome of organisms to give organisms new functions

Question 13

what can we learn from genome wise comparisons between individuals of different species
6.1.3(b)

Answer 13

evolutionary relationships

Question 14

what can we learn from genome wise comparisons between individuals of same species
6.1.3(b)

Answer 14

if particular alleles are associated with certain phenotypes or diseases

Question 15

in DNA proffiling what DNA is used and why
6.1.3(c)

Answer 15

non-coding DNA as it contains sequences called short tandem repeats which are short repetitive segments of DNA that can be repeated many times in a long sequence.
Each individual has a different, random number of these repeats

Question 16

how is DNA digested
6.1.3(c)

Answer 16

by restriction enzymes this hydrolyses the DNA at specific base sequences called recognition sequences. This cuts the DNA into fragments

Question 17

how are DNA fragments seperated and how do fragments travel depending on size
6.1.3(c)

Answer 17

by gel electrophoresis larger fragments travel less far along the gel

Question 18

how is the gel stained
6.1.3(c)

Answer 18

the gel is stained with a blue fluorescent dye and a banding pattern can be seen

Question 19

which banding patterns of different DNA samples can be compared
6.1.3(c)

Answer 19

banding patterns of different DNA samples that were cut with the same restriction enzyme can be compared and conclusions can be drawn

Question 20

state 2 applications of DNA profiling
6.1.3(c)

Answer 20

-can be used in forensic science to match suspects with DNA evidence that has been found at the scene of the crime
-in maternity and paternity disputes

Question 21

what is PCR used for
6.1.3(d)

Answer 21

amplify a small quantity of DNA to produce larger quantities

Question 22

what reagents does PCR require
6.1.3(d)

Answer 22

-target DNA to be amplified
-primers
-DNA polymerase (Taq polymerase)
-free DNA nucleotides
-buffer solution

Question 23

what are primers
6.1.3(d)

Answer 23

short sections of DNA
Anneal to complementary bases at the start of the DNA fragment
Allow DNA nucleotides to be added

Question 24

why do we use Taq polymerase
6.1.3(d)

Answer 24

-has a high optimum temperature so wont be Denatured when seperating H bonds

Question 25

why do we use buffer solution
6.1.3(d)

Answer 25

to provide optiumum pH for the reactions

Question 26

what is the number of molecules at the end of the PCR cycle
6.1.3(d)

Answer 26

(initial number of molecules) X 2^(number of cycles)

Question 27

what does PCR take place inside of
6.1.3(d)

Answer 27

thermal cycler-cycles the reaction mixture through set temperature and holds the mixture at the set temperature for an appropriate amount of time

Question 28

what are the 3 stages of PCR
6.1.3(d)

Answer 28

denaturation
annealing
elongation

Question 29

what is denaturation
6.1.3(d)

Answer 29

double stranded DNA is heated to 95 which breaks H bonds holding the DNA strands together

Question 30

what is annealing
6.1.3(d)

Answer 30

temperature is decreases to 50-60 so primers can anneal to the ends of the single DNA strands

Question 31

what is elongation
6.1.3(d)

Answer 31

-temperature is increased to 72 for at least a min
this is optimum temperature for Taq polymerase
adds complementary free nucleotides which and catalyses the formation of phosphodiester bonds between them.

Question 32

what happens after elongation
HAVE TO HAVE THIS POINT
6.1.3(d)

Answer 32

the process is repeated several times to amplify the DNA fragment

Question 33

what is electrophoresis used for
6.1.3(e)

Answer 33

separating different sized fragments of DNA

Question 34

what does the gel electrophoresis tank use
6.1.3(e)

Answer 34

agarose gel covered by buffer solution

Question 35

where are electrodes placed
6.1.3(e)

Answer 35

at each end of the tank and connected to the power supply so an electric current can pass through the buffer solution

Question 36

why can the electric current pass through the buffer
6.1.3(e)

Answer 36

contains ions so is conductive

Question 37

why do all DNA molecules have a negative charge
6.1.3(e)

Answer 37

due to the phosphate group

Question 38

what happens to larger DNA molecules
6.1.3(e)

Answer 38

do not travel as far through the gel

Question 39

what happens to the buffer solution
6.1.3(e)

Answer 39

pour the buffer solution away and add DNA-binding fluorescent tag to the agarose which allows you to see the pattern of fragment

Question 40

how do you identify the DNA fragments
6.1.3(e)

Answer 40

southern blotting
-transfer them onto a membrane made of nitrocellulose
-fluorescent probes that are complementary to a specific base sequences are applied to the membrane. If base sequence of interest is present probe will anneal to it and band will become visible under UV light

Question 41

how do you separate proteins
6.1.3(e)

Answer 41

using gel electrophoresis
however, you use the negatively charged detergent SDS

Question 42

how do you identify the sequence of specific proteins
6.1.3(e)

Answer 42

western blotting-separated proteins must be applied to the membrane. Antibodies specific to proteins of interest are applied. This allows proteins of interest to be visualised

Question 43

describe the process of genetic engineering
6.1.3(f)

Answer 43

1. Restriction endonucleases are used to cut the desired gene from the DNA
   
   2. This creates sticky ends which make it easier to insert the desired gene
   3. The plasmid (vector) is cut using the same restriction enzyme to produce complementary sticky ends.
   4. The desired gene is inserted into the plasmid using DNA ligase which catalyses the formation of phosphodiester bonds between the nucleotides of the desired gene and plasmid. A recombinant plasmid will be formed
   5. The desired gene is inserted along with a marker gene eg-fluorescent marker gene or antibiotic resistance marker gene.
   6. The recombinant plasmid is inserted into the host cell using Ca2+ and heat shock or electroporation. This is where the an electric shock makes the membrane more porous and therefore plasmids can pass through the membrane of the host cell.
      If bacteria has successfully take up the recombinant plasmid they would express the marker gene. So they would be able to glow under UV or would survive when antibiotics are added.

Question 44

what is recombinant DNA
6.1.3(f)

Answer 44

DNA that is combined from 2 sources

Question 45

what is a negative of using antibiotic resistance genes as marker genes
6.1.3(f)

Answer 45

could lead to antibiotic resistance spreading to the wider population
potentially pathogenic bacteria

Question 46

negatives of GM soya seeds
6.1.3(f)

Answer 46

soya beans have been GM to produce a bacterial toxin called BT that kills insects however it could potentially be toxic to other insects

Question 47

negatives of crop plants being GM to produce the herbicide glyphosphate
6.1.3(g)

Answer 47

this means glyphosphate can be sprayed on plants without killing desired crops only weeds increasing yield of crops
however poorer farmers don’t see the same level of profit as they cant afford GM crops

Question 48

using mice for cancer treatment
6.1.3(f)

Answer 48

mice can be GE to carry mutant alleles of genes knows to be linked to cancer which we can use for research
however there’s ethical issues with using animals for research

Question 49

GM goats and sheep
6.1.3(f)

Answer 49

goats and sheep can be GM to produce useful proteins in there milk
issues with using animals

Question 50

negative issues with patenting
6.1.3(f)

Answer 50

patenting prevents people in less economically developed countries from buying GM
they also rely on havesting seeds from 1y to the next. Something which patenting may not make possible

Question 51

what is gene therapy
6.1.3(h)

Answer 51

modifying genes in a patients cells to treat genetic disease
the basic principle is that you replace a faulty allele with a functional one

Question 52

what is somatic cell gene therapy
6.1.3(h)

Answer 52

genetic modification is made to non-reproductive body cells
so it will only affect the patient

Question 53

what is a negative of somatic cell gene therapy
6.1.3(h)

Answer 53

somatic cell eventually die so the treatment will have to be repeated

Question 54

what is germline cell gene therapy
6.1.3(h)

Answer 54

genetic modification made to gametes
this means changes will be passed into future generations

Question 55

what is some ethical issues surrounding germline cell gene therapy
6.1.3(h)

Answer 55

-unborn children cant consent to being genetically modified
-effects of gene therapy may be unpredictable and potentially negative