6.1.3 manipulating genomes

Question 1

what is a genome?

Answer 1

all of an organisms genes

Question 2

what does DNA sequencing determine?

Answer 2

order of nucleotides in a DNA sample

Question 3

what is the purpose of PCR?

Answer 3

to amplify DNA so you can find out the order of bases

Question 4

why is PCR used?

Answer 4

• DNA profiling (forensics)
• finding cancerous mutations
• finding out what genes do (characterization)

Question 5

describe DNA fragmentation

Answer 5

1. before PCR, DNA cut into fragments
2. palindromic sequences in DNA recognized by restriction endonucleases which cut/ digest the DNA at these sites
3. leaving “sticky ends” that overhang
4. each restriction enzyme is specific to a sequence and comp to its active site

Question 6

state the 4 stages of PCR

Answer 6

1. denaturation
2. annealing
3. elongation
4. amplification

Question 7

describe stage 1 (denaturation) of PCR

Answer 7

• DNA heated to 94-6 degrees
• 15-20 secs
• DNA separates into single strands

Question 8

describe stage 2 (annealing) of PCR

Answer 8

• cool mixture to 68
• primers anneal to DNA by comp base pairing

Question 9

describe stage 3 (elongation) of PCR

Answer 9

• heat to 72
• let DNA (taq) polymerase to extend primers
• copies of target DNA synthesized using target strands as templates

Question 10

why does the genome need to be fragmented before sequencing?

Answer 10

• genome is too big
• smaller fragments have fewer errors
• more accurate
• otherwise would take too long

Question 11

compare PCR and in vivo gene cloning

Answer 11

1. PCR - quicker, takes few hours
   in vivo- weeks
2. PCR - less equipment needed - tube and heat
   in vivo- multiple tubes and agar plates
3. PCR- easier and less costly (set to run and left)
   in vivo - have to pick out and transfer the colonies
4. PCR - combines selection of DNA and amplification (primer selects only target gene to be copied)
   in vivo- needs probe to correctly identify the gene

Question 12

5 stages of DNA profiling

Answer 12

1. extraction of DNA
2. digesting sample
3. electrophoresis
4. hybridization
5. evidence

Question 13

explain how the locations of the fluorescent on DNA reveal which genes are now active

Answer 13

• mRNA binds to gene probes by comp base pairing
• the more active the gene the more mRNA produced
• during transcription
• so more fluorescent indicates more mRNA

Question 14

stages of genetic engineering

Answer 14

1. the gene of interest is cut out from the DNA using restriction enzymes
2. the gene is inserted into a plasmid (vector) using DNA ligase and into bacteria
3. bacteria that contain new DNA are called transgenic bacteria
4. they are grown on agar plates containing antibiotics so you can distinguish them from bacteria that have not taken up any new DNA
5. a fluorescent probe can be used to identify bacteria that have desired sequence of DNA

Question 15

explain how the enzyme DNA ligase is used

Answer 15

• used to join pieces of DNA together to make recombinant DNA (DNA that is joined together from different organisms)
• reseals cut ends of DNA
• forms the sugar phosphate backbone

Question 16

what is reverse transcriptase enzyme?

Answer 16

RNA made into DNA (reverse of transcription)

Question 17

outline the procedure by which bacteria can be genetically modified to produce renin.

Answer 17

1. cut out renin gene using restriction enzyme
2. if you don’t know the gene sequence - extract mRNA and use reverse transcriptase to make ss cDNA and use DNA polymerase to make it ds
3. cut the bacterial plasmid with restriction enzymes, leaves sticky ends
4. cut the rennin gene with same restriction enzyme leaving comp sticky ends
5. use DNA ligase enzyme to anneal and join sugar phosphate backbones to form recombinant DNA
6. insert the vector into the cell using heat shock (with CaCl2) or electroporation
7. Transformation used to identify when cells have taken up vector containing new gene

Question 18

suggest 2 ways which bacteria that take up modified plasmid can be identified.

Answer 18

• fluorescent marker ( marker gene) bacteria that fluoresce under UV light have plasmid
• insert antibiotic resistant genes into new genes
• grow on agar containing antibiotics
• survivors will have the modified plasmid

Question 19

describe somatic gene therapy

Answer 19

• replacing mutant gene with healthy allele
• not passed onto offspring
• involves liposomes ( artificial vesicles that package the DNA and fuse with the cell membrane to deliver DNA to target cells)
• normal body cells

Question 20

describe germ line gene therapy

Answer 20

• inserting healthy allele into germ allele
• usually egg
• can be sperm or fertilized egg
• differentiation and growth occurs
• added allele present in every cell of body
• passed onto offspring via gametes

Question 21

what is gene therapy?

Answer 21

treating generic disease by changing natural genotype

Question 22

describe a difference between somatic and germ line gene therapy

Answer 22

somatic targets specific tissues in need of treatments germ line doesn’t

Question 23

suggest 3 developments that have led to an increase in the speed at which DNA can be sequenced

Answer 23

1. high throughput sequencing
2. shotgun sequencing
3. pyrosequencing

Question 24

differences between the base G (in DNA)
and an ATP molecule

Answer 24

G base contains guanine
ATP contains adenine

G has 1 phosphate
ATP has 3 phosphates

G contains deoxyribose
ATP contains ribose

In G phosphate it attached to C3
In ATP no phosphate attached to C3

Question 25

how does DNA sequencing allow the sequence of amino acids in a polypeptide to be predicted?

Answer 25

• used to determine order of bases
• order of bases codes for the sequence of amino acids
• each triplet of bases code for 1 amino acid

Question 26

a virus has a rapid mutation rate so scientists worked to develop a vaccine against it. outline how DNA sequencing and bioinformatics are used to increase the effectiveness of a vaccination programme against the virus.

Answer 26

DNA sequencing
- find the order of bases
- virus has a high mutation rate which leads to many STRAINS being produced
- scientists can predict the viral strain
- so the vaccine contains the correct antigen

Bioinformatics
- uses computer software to analyze store and organize biological data about DNA, RNA and proteins.
- the data is universal and shared internationally
- it can identify the source of the outbreak and vulnerable populations
- so the vaccination programme can target certain areas

Question 27

how can bioinformatics be useful in determining whether a newly sequenced allele causes a genetic disease?

Answer 27

1. • the data base can be used to find the known base sequence of the healthy normal allele
   • computational analysis allows fast comparison with the newly sequenced allele
2. • the a.a sequence in held in the data base
   • computer modeling of new protein structure from data base

Question 28

explain why only selected sections of non-coding DNA are used during DNA profiling in humans

Answer 28

• in most humans the genome is similar / most genes are the same
• using coding DNA would not provide unique profiles
• parts of non-coding DNA contain short tandem repeats (STRs)

Question 29

how do you calculate how many DNA fragments might be present after a certain number of cycles?

Answer 29

• 2^n
• 2 because it is double stranded
• n = number of cycles

Question 30

how many DNA fragments might be present after 12 cycles of PCR. 1 DNA fragment at start. represent answer as log10 value.

Answer 30

• 2^12 = 4096
• log(4096) = 3.61
• answer is 10^3.61

Question 31

why might the number of DNA fragments calculated after 12 cycles not be achieved in practice?

Answer 31

• temperature damages fragment
• template strands could rejoin after being separated rather than primers annealing
• lack of primers
• primers fail to anneal to fragment

Question 32

name of enzyme used in PCR to synthesise new DNA strands

Answer 32

taq polymerase

Question 33

what is the purpose of the buffer/ alkaline solution in gel electrophoresis?

Answer 33

solution carries charge/ current to separate fragments

Question 34

why is taq polymerase used instead of dna polymerase?

Answer 34

• thermostable
• does not denature at 95 degrees when the strands are separated
• so PCR can be cycled repeatedly without stopping to reload the enzyme.

Question 35

describe how DNA can be visualized after electrophoresis.

Answer 35

fluorescent tags which fluoresce under UV light

Question 36

explain how the gene is inserted into a vector

Answer 36

• plasmid cut by restriction enzyme
• leaves sticky ends
• gene cut with same restriction enzyme
• leaves comp sticky ends
• so gene is inserted into vector by comp base pairing
• using DNA ligase

Question 37

how do you obtain the gene of interest if you don’t already know the gene sequence?

Answer 37

• make single stranded complementary DNA
• using enzyme reverse transcriptase
• use DNA polymerase to make double stranded DNA

Question 38

4 differences between somatic and germ line gene therapy

Answer 38

1. somatic - genes not inherited / passed onto offspring
   germ line - genes inherited
2. somatic - gene introduced into body
   germ line - gene introduced into egg/ sperm cell
3. somatic - only some cells get functional gene
   germ line - all cells get functional gene
4. somatic - short term
   germ line - long term

Question 39

ethical concern of testing on animals

Question 40

arguments for and against animal testing

Answer 40

for -
1. animal closely related to human
2. helpful to human health
3. both primates

against -
1. can cause physical harm
2. animal has no choice
3. animal suffers