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21 recombinant DNA technology Flashcards

1
Q

define genome

A

the complete set of genes in a cell of an organism

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2
Q

what is happening to the way we sequence genomes?

A

constantly improving and advancing
has now become automated

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3
Q

which organism has DNA that doesn’t contain introns?

A

prokaryotic cells

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4
Q

define proteome

A

full set of proteins the genes of an organism can code for

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5
Q

why can’t the genome easily be used to translate the proteome in a human, but in bacteria it can?

A

human DNA has introns
bacterial DNA has no introns

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6
Q

why is it useful to be able to translate the genome of bacteria to its proteome?

A

identifying potential antigens to use in a vaccine

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7
Q

what does recombinant DNA mean?

A

a cell having 2 or more sources of DNA

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8
Q

how can recombinant DNA be achieved?

A

by isolating the fragments of DNA and inserting into another organism

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9
Q

what are 5 steps in recombinant DNA technology?

A
  1. isolation of genes
  2. insertion
  3. transformation
  4. identification
  5. cloning
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10
Q

what are 3 ways to isolate a copy of a specific DNA fragment?

A
  1. reverse transcriptase
  2. restriction enzymes
  3. gene machine
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11
Q

how would reverse transcriptase isolate a copy of a specific gene?

A

1.free DNA nucleotides bind to single stranded mRNA (complementary base pair)
2. reverse transcriptase joins DNA nucleotides together to form single stranded cDNA
3. DNA polymerase makes cDNA double stranded

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12
Q

give 3 advantages of using reverse transcriptase as a way of isolation

A
  1. cDNA intron free
  2. mRNA much easier to obtain
  3. mRNA can be isolated from the cytoplasm of cells which produce the protein in large amounts
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13
Q

what are the restriction enzymes called?

A

restriction endonucleases

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14
Q

how would restriction enzymes isolate a copy of a specific gene?

A
  1. restriction endonuclease hydrolyse DNA at specific recognition sequence
  2. recognition sequence palindromic
  3. cut is either blunt or sticky
  4. blunt ends have no overhang so cannot reanneal
  5. sticky ends have overhang so do reanneal
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15
Q

how would the gene machine isolate a copy of a specific gene?

A
  1. desired sequence fed into computer
  2. synthesis of oligonucleotides
  3. oligonucleotides joined together and made double stranded by PCR
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16
Q

what are oligonucleotides?

A

short sequences of nucleotides

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17
Q

what is an advantage for using the gene machine?

A

artificial genes are easily transcribed and translated by prokaryotes as they have no introns

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18
Q

how do you insert genes into plasmid?

A

through vectors

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19
Q

define vector

A

DNA carrier used to transfer foreign DNA into cells

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20
Q

how do you insert DNA into a vector?

A
  1. cut open vector with same restriction endonuclease
  2. complementary sticky ends
  3. DNA ligase anneals DNA fragment to vector DNA (ligation)
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21
Q

what bond does ligation form?

A

phosphodiester bond

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22
Q

explain transformation

A
  1. DNA vector transferred into bacteria
  2. host cells referred to as recombinant or transformed organism
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23
Q

explain how you can identify transformed host cells

A

add marker genes onto the cells and only transformed host cells will synthesise desired protein

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24
Q

why might some cells not be transformed?

A
  1. plasmid may not have entered host cell
  2. host plasmid with sticky ends may reanneal
  3. DNA fragment could loop and join to itself
25
once the transformed cells are identified, what 2 methods are used to clone?
1. in vivo via bacteria 2. in vitro via PCR
26
how do transformed bacteria cells clone?
1. culture bacteria 2. they reproduce by binary fission to produce genetically identical cell with desired inserted gene 3. organisms will produce large quantities of desired protein
27
describe the process of PCR (6 marks)
1. heat DNA to 95 to break weak H bonds and separate strands 2. add primers and nucleotides 3. cool to 50 to allow binding of nucleotides/primers 4. add DNA polymerase 5. heat to 75 6. DNA polymerase joins nucleotides together 7. repeat
28
what are primers?
short pieces of single stranded DNA with complementary base sequences to bases on DNA fragments you want to isolate
29
what do primers do in PCR?
prevent DNA strands from sticking back together and guide DNA polymerase
30
how do you calculate the number of DNA strands when you have 5 cycles?
2^n so 2^5
31
how do you calculate the number of cycles from the number of DNA strands?
log2^(number of DNA)
32
what are the limiting factors in PCR?
- nucleotides - primers
33
what are the 3 main stages of PCR?
1. separation 2. annealing 3. synthesis
34
briefly outline the process of separation in PCR
heat at 95 to break weak H bonds
35
briefly outline the process of annealing in PCR
cool to 50 and add primers and nucleotides
36
briefly outline the process of synthesis in PCR
heat to 75 to activate DNA polymerase for 2 new DNA fragments
37
compare in vivo and in vitro cloning
IN VIVO: 1. can also be used to produce protein or mRNA 2. cells have mechanisms for correcting errors 3. slower IN VITRO: 1. cells can only copy DNA 2. lacks error correcting mechanisms 3. quicker
38
what are 4 benefits of recombinant DNA technology?
1. developing medical applications 2. develop agricultural applications 3. better understanding of biological processes 4. design new industrial processes
39
what are 4 concerns regarding DNA technology?
1. antibiotic resistance 2. inserting new genes could disrupt other genes 3. weeds could become resistant to herbicides 4. DNA may become concentrated in the wealthy people
40
give an example on how recombinant DNA could help develop medical applications
produce large quantities of insulin to treat t1 diabetes OR gene therapy
41
give an example on how recombinant DNA could help develop agricultural applications
produce crops with larger yields to help meet the rising food demands of human population
42
which biological processes could recombinant DNA technology help better our understanding of?
- gene expression - mitosis - DNA replication
43
what is gene therapy?
DNA technology can be used to insert a gene for a functioning protein into the people who suffer from genetic diseases
44
briefly outline how gene therapy can be used to treat people with cystic fibrosis
cystic fibrosis is caused by a mutated gene that codes for a CFTR protein that does not prevent mucus from lining airways so inserting a gene that could code for a protein that does prevent mucus
45
what are somatic and germ line gene therapies?
somatic- DNA transfer to body tissue germ line- DNA transfer to cells that produce egg/sperm
46
what is the difference between somatic and gene line therapy?
somatic- not heritable gene line- heritable to children
47
what would have to be used, instead of plasmids, to carry genes into human cells?
viruses or liposomes
48
what are liposomes?
lipid droplets which cross the phospholipid bilayer and release target DNA into cell
49
what is a probe?
short single stranded DNA that is complementary to an allele and labelled radioactively or fluorescently
50
briefly explain what a probe does
locates specific alleles of genes to screen patients for things like heritable disases
51
explain how a probe can be used to locate a specific allele
1. sample of patients DNA removed and heated 2. mixed with DNA probes with specifically complementary base pairs to disease 3. DNA will bind to probe if patient has the disease allele 4. wash unattached probes away 5. identify probes with X ray or UV light
52
what are the parts in between genes called?
VNTR
53
what does VNTR stand for
variable number tandem repeats
54
do people have the same VNTRs?
no
55
do you inherit VNTRs from parents?
yes
56
explain the process of DNA fingerprinting
1. DNA extracted from sample 2. restriction endonuclease hydrolyse DNA into small fragments 3. VNTRs stay in tact 4. separation using gel electrophoresis 5. transferred to nylon sheet 6. probe identified using X ray
57
explain the process of gel electrophoresis
1. sample loaded into wells in gel with electrical current ran through 2. soaked in strong alkali to make DNA single stranded
58
why is an electrical current added to the gel in gel electrophoresis?
to allow negatively charged DNA samples to move through the gel towards the positive end
59
which DNA fragments in gel electrophoresis move the furthest and fastest?
smaller fragments

biology (23 decks)

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