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Biology - Mr Kelly > 21.1 - 21.5 Recombinant DNA technology > Flashcards

21.1 - 21.5 Recombinant DNA technology Flashcards

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1
Q

How did the Human Genome Project obtain their data?

A

Whole genome shotgun sequencing:
* Cut DNA into small sections
* Use computer algorithm to align overlapping segments and assemble whole genome

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2
Q

Why is it difficult to work out the proteome from the genome?

A
  • Hard to identify which regions are introns
  • Hard to identify which genes are expressed or not expressed
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3
Q

Define complete proteome

A

All the proteins produced in an organism at a given time under specified conditions

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4
Q

Define cellular proteome

A

All the proteins produced in a given type of cell at a given time under specified conditions

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5
Q

Define recombinant DNA

A

DNA that is made from the DNA of two different organisms

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6
Q

Define transgenic/genetically modified organism

A

An organism that contains DNA from two different organisms

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7
Q

Why does DNA function normally even when inserted into a different organism?

A
  • Genetic code is universal
  • Transcription and translation are similar in different organisms
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8
Q

What are restriction endonucleases?

A
  • Enzymes that cut DNA at recognition sequences
  • They hydrolyse phosphodiester bonds in the middle of the polynucleotide chain
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9
Q

Describe sticky ends and their function

A
  • Ends are cut with short stretches of single-stranded DNA with complementary sequences
  • This means they can anneal to other sticky ends cut with the same restriction endonuclease
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10
Q

Where are restriction enzymes obtained from?

A

Naturally produced by bacteria as a defence against viruses

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11
Q

Describe how reverse transcriptase can be used to make DNA fragments

A
  • Reverse transcriptase synthesises a new strand of DNA, complementary to the mRNA, forming the DNA/RNA molecule
  • The strands separate
  • A second DNA strand is synthesised, complimentary to the first
  • This double stranded DNA molecule is called cDNA
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12
Q

Where is reverse transcriptase obtained from?

A

Produced naturally by retroviruses to help them invade cells

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13
Q

What are the advantages of using reverse transcriptase over restriction endonucleases?

A
  • Doesn’t impact on the DNA of the donor organism
  • Doesn’t contain introns so can be inserted into a prokaryote
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14
Q

Describe how a ‘Gene Machine’ is used to produce a DNA fragment

A
  • The known amino acid sequence is used to work out the mRNA codons and then the DNA triplets
  • A computer is used to assemble the bases as necessary
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15
Q

How must a DNA fragment be prepared for insertion?

A

Add promoter and terminator regions

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16
Q

Define promoter

A

A specific sequence of DNA bases (before the start of a gene) that a transcription factor can bind to, in order to stimulate transcription and allow the gene to be expressed

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17
Q

Define terminator

A

A sequence of DNA bases which triggers the RNA polymerase to be released, ending transcription

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18
Q

Describe how a DNA fragment is inserted into a plasmid

A
  • The same restriction endonuclease used to cut out the DNA fragment, is used to break the plasmid loop
  • This ensures the sticky ends are complimentary
  • DNA ligase is used to join the DNA backbone by forming phosphodiester bonds
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19
Q

Describe the introduction of DNA into a host cell

A
  • The plasmids are reintroduced to their host cells, in a process called transformation
  • Plasmids and bacteria are mixed with calcium ions in an environemnt with a frequently changing temperature to increase the bacterial membrane permiability
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20
Q

Why might the introduction of DNA to the host cell not work?

A
  • Only a few cells take up the plasmid
  • Sometimes plasmids close up before incorporating DNA fragment
  • DNA fragments join together to form a loop
21
Q

Define marker gene

A

A gene that is used to identify if a DNA fragment has been successfully incorporated into a host cell

22
Q

Give three examples of marker genes

A
  • Antibiotic resitance
  • Fluorescent markers
  • Enzyme markers
23
Q

Describe how marker genes may be used to identify if a host cell has taken up the DNA fragment

A
  • Use a plasmid that contains two marker genes, one for antibiotic resistance and one for fluorescence
  • DNA fragment should be inserted into gene for fluorescence
  • Place host cells in antibiotic, only those with the plasmid will survive
  • Check these surviving host cells for fluorescence, those with the DNA fragment will not fluoresce
24
Q

What is PCR?

A
  • Polymerase chain reaction
  • A method of copying fragments of DNA
  • Automated, rapid and effecient
25
Q

List the three stages of PCR

A
  1. Seperation of the DNA strands
  2. Annealing of the primers
  3. Synthesis of DNA
26
Q

What is a thermocycler?

A

Computer controlled machine that varies the temp precisely over a period of time

27
Q

Describe seperation of the DNA strands in PCR

A
  • DNA fragments, primers and DNA polymerase are mixed and placed in the thermocycler
  • The temp is increased to 95 degrees c, breaking the hydrogen bonds between complimentary base pairs and causing the two strands to seperate
28
Q

Describe the annealing of primers in PCR

A
  • Mixture is cooled to 55 degrees c
  • Primers join to their complimentary bases at the end of the DNA fragment
29
Q

Describe the synthesis of DNA in PCR

A
  • Temp increased to 72 degrees C
  • This is the optimum temp for DNA polymerase to add complimentary nucleotides along each of the separated DNA strands, beginning at the primer end
30
Q

Describe the function of primers

A
  • Provide the starting sequences for DNA polymerase to begin DNA copying as DNA polymerase can only attach nucleotides to the end of an existing strand
  • Two different primers are used to prevent the two DNA strands from just rejoining
31
Q

Describe the advantages of in Vitro gene cloning

A
  • Extremely rapid
  • Does not use living cells so no complex culturing techniques are required
32
Q

Describe the advantages of in Vivo gene cloning

A
  • Involves vectors which can be used to deliver the gene to another organism
  • Only a gene cut by the same restriction endonuclease as the plasmid can be taken up so there is no risk of contamination
  • Much fewer errors or mutations
  • Copies specific gene not whole DNA sample
  • Produces transformed bacteria that can be used to produce large quantities of gene products
33
Q

Define DNA probe

A

A short, single-stranded length of DNA that has a label attached to make it easily identifiable

34
Q

Describe how radioactively labelled probes work

A
  • Nucleotides contain the isotope 32P
  • Can be identified using an x-ray film that is exposed by radioactivity
35
Q

Describe how fluorescently labelled probes work

A
  • Emit light under certain conditions
36
Q

Describe how DNA probes can be used to identify particular alleles

A
  • A DNA probe is made that is complementary to the allele
  • The DNA that is being tested is heated to separate the two strands
  • The strands are cooled and mixed with the probe
  • The probe binds to the allele in DNA hybridisation
  • The DNA is washed clean of any unattached probes
37
Q

Describe how a DNA probe is made

A
  • Determine the base sequence of the allele that needs to be located
  • Produce a complimentary DNA fragment
  • Replicate the fragment using PCR
  • Attach markers to the fragments
38
Q

Describe how DNA probes can be used in genetic screening

A
  • Can inform potential parents about the likelihood of having a child with a genetic disorder
  • Can inform people if they have a genetic predisposition to cancer (due to oncogenes or mutated tumour suppressor genes) so they can take preventative measures
39
Q

Describe how DNA probes can be used in personalised medicine

A
  • Some people’s genes may make certain treatments more or less effective
  • Can help identify treatment and dosage
  • Can save money by not overprescribing drugs
40
Q

Describe genetic counselling

A

Giving advice and information about the results of genetic screening

41
Q

Define genetic fingerprinting

A

A diagnostic tool used in forensic science, plant and animal breeding and medical diagnosis

42
Q

Define Variable Number Tandem Repeats (VNTRs)

A
  • Sections of repetitive, non-coding bases
  • The number and length of VNTRs is specific to each individual
  • VNTRs are more likely to have unique mutations than coding sections of DNA
43
Q

Describe the process of obtaining a genetic fingerprint

A
  1. Extract DNA from sample
  2. Restriction endonucleases cut the DNA into fragments
  3. Fragments are seperated using gel electrophoresis
  4. DNA fragments are transferred from the gel to nylon membrane
  5. Radioactively labelled DNA probes are added
  6. Nylon membrane is placed onto x-ray film
  7. Development of x-ray film leaves dark bands where the DNA probes have attached
44
Q

Describe how gel electrophoresis works

A
  • DNA fragments placed on agar gel
  • Voltage applied
  • The more slowly the fragment moves across the cell, the larger it is
45
Q

Describe how DNA fingerprinting can be used in plant and animal breeding

A
  • Prevent inbreeding during breeding programmes
  • Identify organisms with a desirable allele
  • Identify the paternity of an animal (its pedigree)
46
Q

Describe how DNA fingerprinting can be used in medical diagnosis

A
  • Can identify alleles that may cause genetic disease
  • Identify the nature of microbial infections
47
Q

Describe how DNA fingerprinting can be used to identify genetic relationships

A
  • Identify paternity of a child
  • Identify the genetic diversity of a population
48
Q

Describe how DNA can be used in forensic science

A
  • Can help establish whether a suspect was present at a crime scene
49
Q

Describe how scientists could use a radioactively labelled probe to prove the presence of a given gene

A
  • Extract DNA
  • Add restriction endonuclease
  • Separate fragments using electrophoresis
  • Treat DNA to form single strands
  • Probe will bind to gene
  • Use autoradiography to show the bound probe

Biology - Mr Kelly (17 decks)

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