Chapter 21 - Recombinant DNA technology Flashcards Preview

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Flashcards in Chapter 21 - Recombinant DNA technology Deck (46)
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1
Q

what does GMO stand for?

A

genetically modified organism

2
Q

outline the rough sequence of stages involved with gene transfer

A
isolation
insertion
transformation 
identification
growth/cloning
3
Q

how may we produce DNA fragments (3)

A
  • conversion of mRNA to cDNA using reverse transcriptase
  • using restriction endonucleases to cut fragments from DNA
  • creating the gene in a gene machine
4
Q

outline the process of using reverse transcriptase to make cDNA (4)

A
  • a cell that readily produces the protein is selected
  • these cells contain large amounts of relevant mRNA
  • reverse transcriptase is used to make DNA from RNA and this DNA is complimentary (cDNA) assembled using DNA nucleotides
  • to make the second strand of DNA the enzyme DNA polymerase builds up complimentary nucleotides along the first cDNA template
5
Q

where are restriction endonucleases extracted from?

A

specific bacteria which produce RE in order to cut up the DNA fragments of invading viruses

6
Q

what is a sticky end

A

where the DNA is cut in a staggered fashion

7
Q

outline the process by which genes are manufactured using the gene machine (7)

A
  • determine amino acid sequence of the desired protein and DNA which is complimentary to mRNA is worked out
  • the nucleotide base sequence are fed into the computer
  • sequence checked to ensure it meets biosecurity standards and ethical guidelines
  • the compute designs a series of small, overlapping single strands of nucleotides (oligonucleotides) to be assembled into the desired gene
  • using an automated process each oligonucleotide nucleotide is added in the required sequence
  • gene replicated using PCR (contains no introns) whihc also makes it double stranded due to complimentary nature
  • inserted into plasmid for storage (these act as a vector)
8
Q

define in vivo gene cloning

A

transfer of fragments to a host cell using a vector

9
Q

define in vitro cloning

A

using the polymerase chain reaction

10
Q

what is the importance of sticky ends?

A

if the same restriction endonuclease is used to cut DNA then all the fragments will have ends that are complimentary to one another meaning that any single stranded sticky end will be complimentary to any other

11
Q

what steps must be taken to prepare a DNA fragment for insertion?

A
  • must add a promoter region to the start of sequence in order for RNA polymerase to bind and begin transcription
  • must add terminator region to the end in order to stop transcription
12
Q

what is a vector?

A

a carrying unit for DNA used to transport DNA into a host cell

13
Q

how are DNA fragments inserted into vectors?

A

the DNA fragments are mixed with the opened up plasmids and become incorporated due to complimentary sticky ends

14
Q

how is the process of tranformation carried out?

A

modified plasmids are mixed together in a medium containing bacterial cells and calcium ions, Ca and temperature changes contribute making the bacterial membrane permeable and allowing the plasmids in

15
Q

why might not every bacterial cell contain the modified DNA (plasmid)

A
  • only a few bacterial cells take up the plasmid
  • some plasmids will have closed up again without incorporating the DNA fragment
  • sometimes the DNA fragments join together to form their own plasmid
16
Q

how is it determined which bacterial cells have taken up the modified plasmids?

A
  • some plasmids carry genes for resistance to more than one antibiotic
  • bacterial cells which have taken up the plasmid will have resistance to a specific antibiotic and therefore will survive (this test which bacteria have taken up the plasmids)
  • since the DNA fragment has been inserted into one of these sections which codes for resistance to a specific antibiotic, this antibiotic will kill bacteria with the modified plasmid
17
Q

name 3 marker genes which can be used to determine whether a modified plasmid has been taken up by the bacteria

A
  • antibiotic resistance
  • make fluorescent proteins
  • make enzyme which can be identified by process
18
Q

what is the use of replica plating?

A

making multiple separate cultures of the same bacteria so that some can be tested for antibiotic resistance

19
Q

outline how fluorescent markers are used to identify bacteria containing desired DNA fragments

A
  • green fluorescent protein (GFP) has the cloned gene fragment inserted into the centre of GFP
  • any cells which have not taken up the modified plasmid will continue to fluoresce
  • any cells that have taken up the plasmid will no longer fluoresce
20
Q

outline how enzyme markers are used to identify bacteria containing desired DNA fragments

A
  • required gene is transplanted into the centre of lactase gene
  • if a plasmid containing the desired DNA fragment has been taken up by the bacteria then the cell will no longer produce lactase and therefore will not break down lactose
21
Q

what is the PCR?

A

a method of copying fragments of DNA

22
Q

outline the items required by the process of PCR (5)

A
  • the DNA fragment
  • DNA polymerase
  • primers
  • nucleotides
  • thermocycler
23
Q

what are primers?

A

short sequences of DNA that have a set of bases complimentary to those at the end of each of the two DNA fragments

24
Q

outline the 3 stages involved in PCR

A
  • separation of the DNA strand ~ DNA fragments, primers and DNA polymerase placed in thermocycler at 95 degrees
  • addition of (annealing) the primers ~ cooled to 55 degrees and primers anneal to ends of fragments, providing the starting sequence
  • synthesis of DNA ~ increased to 72 degrees (optimum temp for DNA polymerase) it begins at the primer and adds complimentary bases
25
Q

state the advantages of in vitro gene cloning (2)

A
  • it is extremely rapid, makes billions of copies in hours

- it does not require living cells

26
Q

state the advantages of in vivo cloning (5)

A
  • useful when introducing genes to another organism
  • it involves almost no risk of contamination
  • it is very accurate, few errors in DNA
  • it cuts out specific genes
  • it produces transformed bacteria that can be used to produce large quantities of gene products
27
Q

give 5 benefits of recombinant DNA technology (8)

A
  • can be modified to produce a wide range of substances which can treat disease
  • microorganisms can be used to control pollution
  • GM plants can be used to produce a specific substance for extraction
  • GM crops can be engineered for greater financial and environmental advantages
  • GM crops can help prevent disease
  • GM animals can produce drugs cheaper
  • replacing defective genes may be a cure for certain genetic diseases
  • genetic fingerprinting can be used as a forensic science
28
Q

give 5 limitations of recombinant DNA technology (14)

A
  • cant predict the wider consequence for the environment
  • recombinant genes may pass from one type of organism to another
  • manipulation of genes may lead to disruption of pathways within the cell e.g. could this cause new diseases?
  • GM bacteria often have antibiotic resistance
  • cant be sure of future impact on evolution
  • unforeseen financial consequences
  • will it lead to eugenics
  • consequences of the technology getting into the wrong hands
  • if the financial cost of recombinant DNA techonology can be justified for those in poverty
  • genetic fingerprinting could be used to the detriment of justice
  • immoral to tamper with genes? unnatural
  • the human genome project, companies can patent and own genes?
29
Q

what are DNA probes?

A

short, single stranded lengths of DNA that has some label attached making it easily identifiable

30
Q

name 2 types of DNA probe

A
  • radioactively labelled probes

- fluorescently labelled probes

31
Q

outline how DNA probes are used to locate specific sections of DNA

A
  • double stranded DNA separated (through heating to break hydrogen bonds)
  • separated strands are mixed with the probe that is complimentary to the sequence of the gene to be sought
  • as the mixture is cooled, the probe binds to complimentary sections, known as DNA hybridisation
  • the site at which the probe binds is later identified by radioactivity or fluorescence
32
Q

why might it be important to screen couples for genetic diseases which they do not have?

A

they may be carriers of disease which could mean they pass the genes onto their offspring, these offspring could then have the disease

33
Q

what is the importance of genetic screening for personalised medicine?

A
  • provide advice based on genotype
  • save money by not prescribing treatments which arent effective on certain genotypes
  • avoids medicines which could cause harm to some
  • dosages can be optimised for specific genotype for most effectiveness and least risk
34
Q

what is genetic counselling?

A

counselling where advice and information can be given that enable people to make informed health decisions about themselves and their offspring

35
Q

what is genetic fingerprinting?

A

a diagnostic tool used in forensic science and breeding, based upon the principle that (almost) all individuals have a unique genetic code

36
Q

what are non coding bases of DNA also referred to (not introns)

A

VNTRs , variable number tandem repeats

37
Q

what is gel electrophoresis?

A

technique used to separate DNA fragments according to size

38
Q

outline the process of gel electrophoresis (5)

A
  • DNA is extracted from the cells in a sample (if the sample is small use PCR)
  • DNA cut into fragments by the same restriction endonucleases, one which cuts close to but not within the target DNA
  • DNA fragments are separated according to size across the gel and immersed in alkali to separate the strands
  • hybridisation, probes are used to bind to the ends of the VNTRs (different porbes used for different DNA sequences)
  • X-ray film placed over nylon membrane for development, bars show where DNA fragments placed
39
Q

give the names of each step in electropheresis

A
extraction
digestion
separation
transfer to nylon membrane
hybridisation
development
40
Q

how are the results of electrophoresis interpreted?

A
  • first visually observe similarities between samples
  • then if there appears to be a match pass the two samples through an automated scanner which calculates the likelihood that it is a match
41
Q

what are the main uses of genetic fingerprinting? (4)

A
  • genetic relationships and variability
  • forensic science
  • medical diagnosis
  • plant and animal breeding
42
Q

how is DNA fingerprinting used in genetic relationships

A

used to establish paternity as each bar of a child must correspond to a bar of the mother or father

43
Q

how is DNA fingerprinting used in forensic science

A

used to establish who was present at the scene of the crime

44
Q

why might DNA found at the scene of the crime not prove that a suspect was responsible? (3)

A
  • could be DNA of a close relative
  • DNA could be left from another innocent occasion
  • sample may have become contaminated after the crime
45
Q

how is DNA fingerprinting used in medical diagnosis

A

sample of DNA fragment with specific allele for disease compared to those with the disease shows the likelihood of disease onset in patient

46
Q

how is DNA fingerprinting used in plant and animal breeding?

A

used to prevent undesirable inbreeding in conservation programmes and in zoos or identify organisms with a desirable allele of a gene