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Biology Foundation > XABY01 GENE TECHNOLOGY > Flashcards

XABY01 GENE TECHNOLOGY Flashcards

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1
Q

What happens in genetic engineering?

A

genes are taken from one organism (‘foreign DNA’) and inserted into another host organism, altering the genetic make-up of the organism to produce a transgenic organism

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2
Q

What are commonly used as recipient cells during gene transfer?

A

Bacteria and yeast

sometimes phage viruses are used

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3
Q

Why are microorganisms used in gene transfer?

A

Rapid reproduction rate enables transferred gene to be copied so that a large amount of gene product can be obtained

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4
Q

what is the difference between In Vivo cloning and In Vitro cloning?

A

In Vivo = copies made inside a living organism

In Vitro = copies made outside of a living organism

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5
Q

What is used instead of a specific gene or fragment of DNA from an organism in genetic engineering?

A

mRNA that has been transcribed from the gene is removed from the cells and used

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6
Q

what is the process of genetic engineering using reverse transcriptase? (5)

A
  1. mRNA transcribed from the desired gene is isolated from cells
  2. mRNA mixed with free DNA nucleotides and enzyme reverse transcriptase
  3. free DNA nucleotides align next to their complementary bases on mRNA template
  4. reverse transcriptase joins the DNA nucleotides together to produce a cDNA fragment (gene)
  5. double stranded DNA produced from cDNA using DNA polymerase
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7
Q

what is cDNA?

A

complementary DNA, produced by the reverse transcriptase technique

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8
Q

What role do restriction endonuclease enzymes play in GE?

A

they cut the sugar-phosphate backbone and double stranded DNA molecule at a unique, specific base sequence (recognition site)

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9
Q

Most recognition sites are palindromic. What does this mean?

A

they read the same either way on each polynucleotide strand

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10
Q

What do sticky ends do?

A
  • enable DNA to be joined/spliced onto a different piece of DNA more effectively
  • due to staggered cut (overlapping base) that form hydrogen bonds with complementary bases on the other sticky end
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11
Q

why are enzymes that produce blunt ends not preferable?

A

there is more chance of the ends joining non-specifically

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12
Q

How is a gene incorporated into plasmid DNA?

A

using enzyme DNA ligase

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13
Q

What role does DNA ligase have in plasmid genetic engineering?

A
  • rejoins the break between nucleotides of each strand of DNA reforming the sugar phosphate backbone of the plasmid
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14
Q

How is the new recombinant DNA copied?

A

using the host cell’s DNA replication machinery or copied using an artificial process such as PCR

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15
Q

What are the steps in preparation of a recombinant plasmid?

A
  1. a gene of interest (DNA fragment) is isolated from human tissue cells
  2. an appropriate plasmid vector is isolated from a bacterial cell
  3. human DNA and plasmid treated with the same restriction enzyme to produce identical sticky ends
  4. restriction enzyme cuts the plasmid DNA at its single recognition site, disrupting the pre-existing gene present
  5. mix the DNA together and add enzyme DNA ligase to bond stick ends
  6. recombinant plasmid introduced into bacterial cell by simply adding DNA to a bacterial culture where some bacteria take up the plasmid from solution
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16
Q

What must cells be in order to take up DNA?

A

competent (primed to take up DNA more readily)

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17
Q

for bacteria, what does being competent require?

A

a change in culture conditions (a high calcium ion concentration alters membrane properties)

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18
Q

how are plasmids (vectors) taken up by bacterial cells?

A
  • plasmids containing recombinant DNA added to competent bacteria
  • some of which take up plasmid by endocytosis
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19
Q

What is a more efficient alternative method of making bacteria competent?

A

electroporation

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20
Q

how does electroporation work?

A
  • bacteria and plasmids mixed together
  • very high voltage applied for a short period of time
  • transient disruption of cell membrane allows DNA to pass into the cell
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21
Q

What is a bacterial cell said to be if it takes up a plasmid?

A
  • transformed

- now a transgenic organism

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22
Q

what 3 things can be used as vectors to allow plasmids to be taken up by cells/tissues?

A
  1. viruses
  2. liposomes
  3. ballistic projectiles
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23
Q

is transformation of bacteria efficient? why?

A
  • no

- large numbers of bacteria with no plasmids will be present as will bacteria that have no foreign gene

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24
Q

what allows transformed bacteria with recombinant plasmids to be detected?

A

genetic markers

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25
Q

what do marker genes (genetic markers) do?

A

enable genetically engineered bacteria to be detected and isolated for subsequent culturing

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26
Q

what genes is some plasmids can be used as markers?

A

antibiotic-resistance genes

often genes for ampicillin or tetracycline resistance

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27
Q

How are antibiotic-resistance genes used as markers?

A
  • inserted gene will disrupt gene for resistance already present, so resistance is lost
  • transformed bacteria identified as they will be resistant to ampicillin but not tetracycline
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28
Q

why are antibiotic-resistance genes used as markers less nowadays?

A
  • because of the risk of spreading antibiotic resistance by horizontal gene transmission from one species to another
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29
Q

what other markers can be used instead?

A

fluorescent markers

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30
Q

what does the GFP gene code for?

A

production of a green fluorescent protein (GFP)

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31
Q

How is the GFP gene used as a marker?

A
  • cloned gene added to GFP gene

- successfully transformed bacteria can be identified as they fluoresce when viewed with UV light under a microscope

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32
Q

How is a recombinant plasmid replicated?

A
  • any bacteria taking up the recombinant plasmid will replicate it during cell division to produce a colony - clones of genetically engineered bacteria
33
Q

How is the gene product acquired from the genetically engineered colony? (3)

A
  • bacterial colony can be harvested and stored
  • plasmid can be purified, digested with restriction enzymes and analysed using gel electrophoresis to confirm presence/size of foreign DNA
  • bacterial cells cultured and produce gene product in large amounts (e.g. insulin, blood clotting factors etc.)
34
Q

what allows bacteria containing recombinant plasmids to be cultured on a large scale?

A

industrial fermenters

35
Q

what are chemicals produced using large scale culturing?

A
  • enzymes
  • antibiotics
  • hormones
36
Q

How is gene product acquired from large scale industrial fermenters?

A
  • fermenters sterilised before adding liquid medium and genetically engineered bacteria
  • temperature and pH controlled to provide optimum growth conditions
  • bacteria divide and produce gene product, which can be extracted and purified
37
Q

What is electrophoresis?

A

technique used to separate and sometimes purify macromolecules

38
Q

How does electrophoresis work?

A
  • molecules separated by gel matrix of either agarose (polysaccharide from seaweed) or chemical matrix polyacrylamide
  • gel acts as a sieve
  • with larger molecules moving slowly through the gel and smaller ones moving faster
39
Q

what is the process of electrophoresis? (7)

A
  • samples containing DNA mixed with a loading buffer (sucrose and blue dye) and placed into sample wells
  • current is applied (negative at the well end of the gel) and as nucleic acids have a negative charge they migrate through the gel toward the anode (positive electrode)
  • as DNA migrates toward anode, separates according to its size (length)
  • smaller fragments travel more quickly through the gel, larger fragments more likely to get trapped and progress slowed
  • DNA fragments made visible in the gel by staining with a fluorescent dye and placing under a UV light
  • gel photographed to record position and fragments of DNA can be cut and extracted from gel
  • exact size can be calculated by comparison with DNA ladder (DNA of known size)
40
Q

what is the dye used for in electrophoresis?

A
  • dyes in loading buffer show progression of separation
41
Q

what does using gels with different concentrations of agarose allow us to do?

A
  • allows different sizes of DNA fragments to be resolved
  • larger fragments much better resolved in the 0.7% gel
  • small fragments separated best in 1.5% agarose
42
Q

What are gene probes?

A

short single stranded lengths of DNA

43
Q

what are gene probes used for? (3)

A
  • to detect the presence of DNA that contains a particular sequence of bases
  • can be used to identify long lengths of DNA (genes) that contain target sequence within them
  • also to identify mutated sequences, most commonly used in genetic fingerprinting
44
Q

How is the gene probe made?

A
  • usually 15-20 bases long made radioactive using a radio isotope of phosphorus (32P)
  • this is incorporated into the sugar-phosphate backbone of the single strand
45
Q

what does the use of probes rely on?

A
  • target nucleotide sequence being known

- if complementary sequence to target is synthesised, then it will bind to the target via complementary base pairing

46
Q

what was the method used to detect if the probe had bound to the DNA fragments?

A
  • using the blotting technique
47
Q

What does the blotting technique entail?

A
  1. DNA fragments separated by agarose gel electrophoresis
  2. separated DNA fragments blotted onto nitrocellulose paper
  3. nitrocellulose paper with tightly bound DNA removed
  4. labelled DNA probe hybridised to separate DNA
  5. labelled DNA probe hybridised to complementary DNA bands visualised by autoradiography
48
Q

How are the gene probes added to the DNA fragments?

A

gene probes bound to a solid surface and then DNA fragments are added

49
Q

How can probe-sample hybridisation be detected and quantified?

A
  • detection of fluorescently labelled sample sequence
50
Q

in genetic fingerprinting, what is the DNA being analysed?

A

the non-coding DNA in between the genes that make up about 95% of human genome

51
Q

what does RFLP analysis stand for?

A 
RF = restriction fragments, fragments of DNA cut by restriction enzymes
L = length, refers to length of restriction fragments
P = polymorphism, (many shapes), as the lengths of some restriction fragments differ greatly between individuals , due to VNTR's, thus many differing lengths of DNA possible in a population
52
Q

what is a VNTR?

A

variable number tandem repeats

53
Q

why does the DNA analysed accumulate mutations that have no impact on the organism?

A

because the DNA is non-coding

54
Q

what kind of mutations take place?

A

additions or deletions of large sections of DNA rather than single bases

55
Q

why are these sequences called ‘tandem repeats’?

A

because as a result of large sections of DNA being added/deleted, these sequences are repeated many times

56
Q

why are these sequences known as ‘variable number’

A

because there is variation within the population in the number of repeated blocks of the same DNA sequence

57
Q

how many alleles of VNTR’s will each individual have at the same locus on each chromosome?

A

2, one maternal and one paternal

58
Q

What is the procedure of genetic fingerprinting?

A
  1. DNA purified from an individual
  2. restriction enzymes cut at sites flanking the tandem repeat sequences
  3. southern blotting method is carried out
  4. if a single probe is used a maximum of 2 bands may be detected representing 2 different alleles (heterozygous)
    - if one band is seen the individual is homozygous
  5. if number of probes used for different sequences, multiple bands are seen - increases the reliability of data as makes it easier to distinguish between two individuals
  6. autoradiography used to detect the probe position on the Southern blot
  7. unique genetic profile (fingerprint) can be produced
59
Q

how can a parental relationship be identified between an individual being tested and a potential parent?

A
  • each child must inherit one band (allele) from each parent
  • if no band is seen, that individual cannot be the parent
60
Q

What does PCR stand for?

A

Polymerase Chain Reaction

61
Q

what is PCR?

A

technique used to amplify a specific nucleic acid sequence In Vitro in order for other methods to be used to determine size, fingerprint or nucleotide sequence

62
Q

how is PCR used in occupational science?

A
  1. forensic science
    - biological material containing DNA (blood, semen, hair (follicle), cheek cells and bone) can be used to help characterise the victim/criminal using very small amounts of material previously an impossible task
    - police now able to solve old cases using modern methods with stored material
  2. Phylogenetic studies
    - DNA sequence info may be obtained from old/extinct biological specimens allowing evolutionary link to be investigated
  3. screening of blood/blood products
    - blood/products can be screened to detect contamination by viruses such as HIV, Hepatitis, CMV
    - prevents transfusions being contaminated and recipient becoming infected
  4. Patient monitoring
    - success of antiviral treatments in immunocompromised individuals (chemotherapy/AIDS patients) can be followed
    - DNA produced can be used in gene therapy
  5. Genetic Screening
    - lot of information being collected about different diseases, particularly those caused by a mutation of a gene that can be passed to offspring (e.g. sickle cell anaemia, cystic fibrosis)
    - allows people at risk to plan families using available info
    - with results of human genome project, info about a genetic component marking an individual as high/low risk could be determined (cancer, heart disease, MS)
    - many copies of an isolated gene can be prepared (bypassing traditional methods using plasmid vectors and bacteria) to obtain large amounts of genetic material
63
Q

How many stages are there of PCR?

A

4

64
Q

What is stage 1 of PCR? (2 steps)

A

Separate Strands:

  1. reaction mix contains primers (short pieces of single stranded DNA), template DNA, nucleotides and DNA polymerase
  2. mixture heated and kept at 95ºC for 2-5 minutes, hydrogen bonds broken between bases. Two DNA polynucleotide template strands produced
65
Q

what is stage 2 of PCR? (3 steps)

A

Bind Primers:

  1. reaction cooled to 45-64ºC - temperature depends on length and base composition of primers
  2. Primers bind to their target sites of each strand and indicated DNA to be copied
  3. DNA polymerase binds to primer/DNA double stranded portion
66
Q

what is stage 3 of PCR? (4 steps)

A

Duplicate (copy) DNA:

  1. temperature raised to enzyme optimum of 72º (reaction fast due to high temperature)
  2. complementary nucleotides base pair with template strand
  3. DNA polymerase move along and joins nucleotides together forming sugar-phosphate backbone in the new DNA strand
  4. two double stranded molecules produced
67
Q

what is stage 4 of PCR? (2 steps)

A

Repeat:
(this cycle of stages 1-3 is repeated 20-40 times)
1. after each cycle the number of product DNA molecules doubles (exponential increase)
2. reaction is stopped and reaction products analysed by gel electrophoresis

68
Q

why is PCR effective?

A

makes many identical copies of original DNA in a short period of time due to exponential increase

69
Q

What is DNA sequencing?

A

a technique used to determine the sequence of nucleotides in a DNA sample

70
Q

How might have the DNA been prepared to be sequenced?

A
  • identified and obtained from Southern blot
  • inserted into plasmid
  • plasmid inserted into bacteria to allow replication
  • plasmid purified and used as a source of DNA to be sequenced
71
Q

what is the Sanger procedure?

A
  • the most common DNA sequencing technique

- requires 4 sequencing reactions to be carried out at the same time

72
Q

what is the difference between PCR and DNA sequencing in regards to amount of DNA needed?

A

PCR can use very small amounts of DNA

Sequencing needs relatively large amount of DNA

73
Q

what will a DNA sequencing reaction tube contain?

A
  1. large quantities of DNA (acting as a template)
  2. DNA primers (similar to PCR except radioactively labelled with 32P)
  3. Normal copies of all 4 DNA nucleotides (ACTG)
  4. DNA polymerase enzyme
  5. Terminator nucleotides
74
Q

What are primer molecules used for in sequencing?

A

used to start the sequencing (provides a double stranded portion of DNA for the DNA polymerase to attach to)

75
Q

What does the radioactivity of the primers do?

A

allows presence of primer to be detected
(an alternative method is that 32P containing nucleotides incorporated into newly synthesised DNA, and primer can be non-radioactive)

76
Q

What do the normal copies of the 4 DNA nucleotides do in sequencing?

A

align via complementary base pairing next to single stranded template DNA

77
Q

What does DNA polymerase do in sequencing?

A

joins nucleotides together forming sugar phosphate backbone, producing new strand of DNA

78
Q

what do the terminator nucleotides do in sequencing?

A
  • small percentage of each of the 4 DNA nucleotides is modified
  • acts as a terminator nucleotide in extending DNA strand
  • nucleotide is a dideoxy nucleotide
  • normal DNA nucleotide is deoxyribose and DNA polymerase can only synthesise DNA in the 5’ to 3’ direction
  • dideoxy nucleotide has a 5’ OH that can attach to growing nucleotide strand, but it does not have an OH group attached to the carbon 3’
  • as a result it cannot be extended further ad will terminate the copying of a DNA strand
79
Q

what is the reaction procedure of DNA sequencing?

A
  1. DNA polymerase uses radioactive primer to begin formation of a complementary DNA strand
  2. DNA nucleotides joined together using sample DNA as a template
  3. random addition of chemically modified nucleotide stops the synthesis of DNA strand; majority of reactions continue and may be terminated later
  4. as a result of random termination a single tube will contain a set of different sized DNA fragments produced as DNA molecules are terminated by A,C,G or T (depending on dideoxy used) at different positions
    NB: all fragments are radioactive due to presence of primer

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