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Biology A2 > Recombinant DNA Technology > Flashcards

Recombinant DNA Technology Flashcards

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1
Q

Suggest why it is possible to transfer DNA from one organism to another

A
  • genetic code is universal

- transcription/ translation mechanisms are universal

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2
Q

Transgenic

A

genetically modified organism

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3
Q

State methods of gene isolation

A
  • restriction endonucleases
  • reverse transcriptase
  • gene machine
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4
Q

Describe how genes are isolated using restriction endonucleases

A
  • active site binds to complementary palindromic recognition site
  • cuts by hydrolysis of phosphodiester bond
  • staggered cut produces sticky ends
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5
Q

Describe how genes are isolated using reverse transcriptase

A
  • reverse transcriptase binds to primer on mRNA
  • transcription occurs with mRNA acting as template to form cDNA
  • DNA polymerase copies cDNA strand forming double-stranded cDNA
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6
Q

Describe how a gene machine is used to artificially synthesise genes

A
  • computer designs oligonucleotides (short strands of nucleotides) using known base sequence
  • DNA polymerase joins oligonucleotides to form desired gene
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7
Q

Evaluate use of reverse transcriptase to isolate genes

A
  • mRNA abundant in cytoplasm
  • no introns so can be transcribed by prokaryotic cells
  • involves multiple steps so time consuming
  • mRNA technically difficult to extract from cytoplasm
  • no regulatory genes
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8
Q

Evaluate use of restriction endonuclease to isolate genes

A
  • create sticky ends to easy to insert into plasmid
  • isolated DNA contains introns so cannot be transcribed by prokaryotic cells
  • difficult to find gene of interest since exact locus must be known
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9
Q

Evaluate use of gene machine to isolate genes

A
  • ability to create any gene
  • no introns so can be transcribed by prokaryotic cells
  • requires specialist equipment
  • must know exact sequence
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10
Q

Sticky End

A
  • formed by restriction enzymes at a palindromic sequence
  • exposed nucleotides at end of DNA fragment
  • complementary to each other
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11
Q

Describe how to prepare a culture of transformed host cells as an in vivo method to amplify DNA fragments

A
  • cut desired gene from cell using restriction endonuclease
  • cut plasmid with same restriction endonuclease
  • (add promoter and terminator regions to fragments of DNA)
  • use DNA ligase to join
  • sticky ends attach
  • use Ca2+ and heat shock
  • plasmid enters bacterial cells
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12
Q

Describe the process of transformation in gene transfer

A
  • medium containing Ca2+
  • heat/electric shock
  • membrane permeable so plasmids enter
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13
Q

Suggest why bacteria cells do not always take up desired gene

A
  • not all plasmids enter bacterial cells
  • some plasmids close up without incorporating DNA
  • some DNA fragments join up to form plasmids
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14
Q

Maker Genes with examples

A

genes used to identify cells that have taken up desired gene, e.g. antibiotic resistance, fluorescent, enzyme

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15
Q

Replica Plating

A

technique to transfer microorganisms from a master plate to a number of further plates in order to classify bacterial colonies

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16
Q

Suggest an advantage of fluorescent markers over antibiotic resistant

A
  • obtain results quickly and easily (does not require replica plating)
  • antibiotic destroys cells containing required gene
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17
Q

Transformation Efficiency

A

total of transformed colonies/mass of DNA available

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18
Q

Explain how sticky ends join up

A
  • unpaired bases

- join by complementary base pairing

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19
Q

Recombinant DNA

A

sections of DNA from more than one TYPE of organism/ species

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20
Q

Suggest how cells can be identified using antibiotic resistance marker genes

A
  • expose to antibiotic

- only resistant survive

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21
Q

Gene Therapy

A
  • introduction of healthy genes

- replacement or inactivation of defective genes

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22
Q

Suggest what kinds of diseases are not suitable for gene therapy

A
  • involve several gene

- affected by environmental factors

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23
Q

Suggest how viral vectors are modified

A
  • remove/ inactivate genes which allow them to replicate

- cannot infect non-target cells

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24
Q

In vitro / Ex vivo

A

cells modified outside the body and transplanted back

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25
Q

In vivo

A

cells modified while still inside the body using vectors which transfer desired gene

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26
Q

Advantages of gene therapy in livestock farming

A
  • large scale manufacture of proteins to treat disease / drugs
  • decrease livestock disease
  • improved efficiency would reduce environmental impact of farming
  • ability to produce alternatives to petroleum based products
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27
Q

Disadvantages of gene therapy in livestock farming

A
  • potential to cause suffering due to random nature of gene insertion so cannot control expression/ modifying embryos can result in birth defects
  • retrovirus use can result in creation of new viruses
  • low success rate
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28
Q

Advantages of gene therapy in agriculture

A
  • improves quality of crops
  • higher crop yield since less susceptible to disease and pest
  • reduced need for pesticides so no pollution = better for environment
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29
Q

Disadvantages of gene therapy in agriculture

A
  • potentially hazardous effects of uncontrolled cross breeding with wild type crops
  • overuse could result in resistant weeds / pests
  • potential to negatively effect animal populations / food chain
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30
Q

Advantages of gene therapy in medicine

A
  • ability to replace defective cells and eradicate disease

- better alternative to current treatments such as blood/bone marrow transplants which has risk of rejection etc.

31
Q

Disadvantages of gene therapy in medicine

A
  • low success rate due to random nature of gene insertion
  • potentially harmful side effects
  • against religious, cultural beliefs - ‘playing God’
  • unknown long term effects of modifying human genome
32
Q

Polymerase Chain Reaction (In Vitro Cloning)

A

technique use to amplify (copy) DNA fragments

33
Q

Describe how DNA is amplified by in vitro cloning

A
  • heat DNA template to 95 degrees C
  • DNA strands denature and separate (H bonds break)
  • cooled to 55 degrees C to allow primers to anneal by complementary base pairing to DNA template
  • free nucleotides bind to DNA template
  • increase temperature to 72 degrees C for DNA polymerisation using Taq polymerase (optimum)
  • cycle repeats several times
34
Q

Suggest why enzymes used in PCR are obtained from bacteria found in hot springs

A

will not denature at high temperatures

35
Q

Primer

A

short sequences of bases complementary to one end of DNA

36
Q

Suggest why primers are used in PCR

A
  • provide starting sequences for Taq polymerase to bind to

- prevent separate strands of DNA from re-joining

37
Q

Evaluate in vitro cloning

A
  • extremely rapid at amplifying small amounts of DNA
  • do not require living cells so no culturing needed which takes time and effort
  • contaminant DNA multiplied
  • inaccurate
38
Q

Evaluate in vivo cloning

A
  • living cells need to cultured which takes time and effort
  • many steps involved
  • no risk of contamination since gene cut by same restriction endonuclease
  • very accurate since mutations are rare and there are mechanisms to correct errors
  • only specific gene cloned not entire sample
39
Q

Describe the effect of contaminated DNA in a sample prior to PCR

A
  • contaminant DNA amplified

- gives false result

40
Q

Suggest situations where only a very small amount of DNA samples may be available for sampling and PCR can be used

A
  • crime scene
  • extinct organism
  • archaeological site
41
Q

Explain why individuals treated with gene therapy do not pass new gene onto their offspring

A

gene is not present in gametes

42
Q

Evaluate the use of viral vectors

A
  • target specific cell types
  • can enter/inject DNA into cells
  • RNA must be converted to DNA
  • can cause immune response
43
Q

Vector

A
  • carries foreign DNA into host cell

- no benefit to carrier

44
Q

DNA probe

A
  • short, single stranded strands of DNA
  • complementary to gene of interest
  • radioactively or fluorescently labelled
45
Q

DNA hybridisation

A
  • section of DNA or RNA

- combines with complementary single strand of DNA

46
Q

Describe the process of genetic screening

A
  • sequence of nucleotides determined by genetic sequencing or from genetic library
  • complementary DNA probe synthesised which is fluorescently labelled
  • PCR to amplify DNA probes
  • probe added to DNA fragments
  • DNA hybridisation where probe binds
  • detected using specific wavelength of light
47
Q

Give applications of genetic screening

A
  • diagnose disease
  • genetic factors which increase chances of disease
  • personalised medicine to determine correct dosages
  • genetic factors which could be passed onto children
48
Q

Evaluate genetic screening of embryos

A
  • parents can terminate pregnancy
  • parents can prepare for needs of child with disorder
  • playing God by selecting desirable traits
  • risk to embryo
49
Q

Evaluate genetic screening of adults

A
  • early diagnosis so higher success rate for treatment
  • take precautions to prevent onset of disease
  • decide if treatments will be successful to save costs
  • determine dosages to avoid over prescription
50
Q

Gel Electrophoresis

A

separating DNA fragments according to size and charge to create a genetic fingerprint

51
Q

Suggest why DNA moves towards positive terminal

A

negatively charged due to phosphate backbone

52
Q

Suggest importance of tracking dye used in gel electrophoresis

A
  • DNA becomes visible

- current is switched off before DNA runs off end of gel

53
Q

Describe the process of genetic fingerprinting

A
  • extraction of DNA and amplify via PCR
  • cut into fragments using restriction endonuclease
  • separate DNA fragments by (gel) electrophoresis
  • treat with alkali to split double helix to single strands
  • transfer DNA fragments from gel onto nylon membrane (southern blotting)
  • DNA hybridised by adding radioactive DNA probes
  • autoradiograph produced from radioactive decay of probes detected by x-ray film
54
Q

Suggest why DNA fragments are digested by same restriction endonucleases before gel electrophoresis

A
  • same restriction endonuclease used to cut at same restriction sites
  • produces small fragments which can be separated (long strands would take too long to separate)
55
Q

Suggest why DNA fragments are submerged in an alkaline solution during gel electrophoresis

A
  • separate double strands of DNA into single strands

- allows DNA probes to bind

56
Q

Suggest why genetic fingerprints can be used to identify individuals

A
  • variable number tandem repeats/ mini satellites
  • all individuals have different VNTRs/ probability two individuals having same VNTRs is very low
  • size of DNA is different so unique genetic fingerprint
57
Q

Variable Number Tandem Repeats

A

repetitive, non-coding bases of DNA

58
Q

Give applications of genetic fingerprinting

A
  • paternity testing
  • measuring genetic diversity
  • forensic science
  • proving pedigree for animal breed
59
Q

Suggest why genetically modified embryos may not survive

A
  • insertion of gene damages DNA / expression

- foreign antigens attacked by immune system

60
Q

Suggest why electrophoresis of DNA from an individual may show one band rather than two

A

Homozygous

61
Q

Suggest how electrophoresis could be used to estimate the number of base pairs in the separated fragments

A
  • compare distance moved by bands with known lengths / sizes

- smaller fragments move further

62
Q

Suggest how scientists could determine if DNA has been fully digested by adding together lengths of DNA fragments

A
  • sum of strands is more than original length of DNA

- indicated undigested strand is added to total so not completely digested

63
Q

Suggest reasons PCR cycle may level out

A
  • nucleotides/ primers exhausted

- enzymes denature

64
Q

DNA polymerase

A

Joins nucleotides

65
Q

Suggest why bacteria are used over tissue cultures to produce genetically modified products

A
  • fast replication so large production in short time
  • easy to culture and extract product from culture
  • bacteria are less prone to infection / contamination
66
Q

Suggest why animals are used over bacteria in the production of some genetically modified products

A
  • bacteria only survive for a short time

- animals can survive for years

67
Q

Suggest how a human gene is usually obtained in gene technology

A
  • mRNA extracted from human cells

- treated with reverse transcriptase to produce cDNA

68
Q

Suggest advantages of using bacteria to synthesise human gene products

A
  • reproduce rapidly to give a large amount of product in a short time
  • product can be extracted easily from culture medium
69
Q

Suggest ethical considerations for gene technology in crops

A
  • duty to feed whole population
  • risk to food chain / ecosystem
  • resistant weeds/ insects could be harmful to humans
70
Q

Suggest how scientists could identify the which gene produces a particular protein

A
  • identify sequence of amino acids in protein
  • use genetic code to identify the codons/base sequences
  • make complementary radioactive gene probe
  • to find those codons in the DNA
71
Q

Suggest why restriction enzymes cut at palindromic sequences

A
  • same sequence in opposite directions at binding site

- restriction enzyme cuts both strands at SAME site

72
Q

Suggest why bioinformatics of fossils is important

A
  • use database to compare common genes in contemporary and extinct organisms
  • understand evolutionary relationships
  • predict functions of genes
73
Q

Suggest why it is most difficult to translate the genome of more complex organisms into the proteome

A
  • introns (non coding DNA)

- regulatory genes

Biology A2 (15 decks)

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