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Biology A2 content > 3.8.4 Gene Technologies > Flashcards

3.8.4 Gene Technologies Flashcards

1
Q

how is reverse transcriptase used to copy a gene?

A

reverse transcriptase taken from retroviruses (HIV) and used to convert mRNA to cDNA. cDNA converted to the double stranded DNA by DNA polymerase.

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2
Q

how do restriction endonucleases/ enzymes work?

A

have an active site complimentary to certain recognition sites on DNA sequence. Cuts DNA at these recognition sites leaving sticky ends.

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3
Q

why are sticky ends useful?

A

sticky ends have unpaired bases which are complimentary to DNA in the vector (another piece of DNA) this is because the same restriction enzymes/endonucleases were used

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4
Q

what are sticky ends?

A

sequence of nucleotides which had been cut from DNA by restriction enzyme / endonucleases

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5
Q

what are the three ways of obtaining a copy of a gene sequence?

A
  • reverse transcriptase (+ DNA polymerase)
  • restriction enzymes / endonucleases
  • gene machine
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6
Q

what is DNA ligase?

A

an enzyme which can join the phosphate sugar framework of two sections of DNA eg. joining sticky ends

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7
Q

what are restriction endonucleases/enzymes?

A

an enzyme from bacteria that can recognise specific base sequences in DNA and cut the DNA at that site

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8
Q

what are the stages in gene cloning?

A

isolation of genes
insertion of genes into vectors
transformation of recombinant DNA into host cells
identification of host cells successfully taken up recombinant DNA
cloning or growth of cells producing the product

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9
Q

what is DNA formed by reverse transcriptase action on mRNA called?

A

cDNA (complementary DNA)

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10
Q

what enzyme is used to convert cDNA to double stranded DNA?

A

DNA polymerase

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11
Q

where do we obtain the reverse transcriptase used for gene cloning?

A

retroviruses (eg.HIV)

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12
Q

where do we get restriction enzymes/endonucleases from?

A

bacteria

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13
Q

what are the benefits of gene machines?

A
  • quick
  • great accuracy
  • DNA is free of introns
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14
Q

what is a transformed cell?

A

a cell with recombinant DNA

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15
Q

why is using mRNA better than DNA?

A

mRNA doesn’t contain introns

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16
Q

what is a vector?

A

anything that can carry DNA into a host organism eg. plasmids

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17
Q

how does transformation occur?

A

host cells and vectors eg. bacteria cells and plasmids are mixed with calcium ions. This makes the cell membrane more permeable. Then a heat/electric shock is used so plasmids can now pass through the membrane easily into the cell.

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18
Q

what happens during identification?

A

recombinant (plasmid) with (ampicillin) resistance gene is cut using restriction enzymes - same ones used to isolate the gene.
DNA ligase used to join gene to (plasmid)
transform bacteria
transfer bacteria to agar plate containing (ampicillin) - tells us whether bacteria has taken in ANY plasmids.

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19
Q

why use identification?

A

not all of the bacteria will contain recombinant DNA. We need to identify the ones that do - can be as few as 1%.

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20
Q

what are the issues with identification?

A

some plasmids close up before gene can be incorporated
leaves some bacteria with normal plasmid and some with altered plasmid

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21
Q

how does two gene markers work for identification?

A

one gene codes for (ampicillin) resistance and the other for (tetracycline) resistance
restriction enzyme used to cut into (tetracycline) resistance gene
DNA ligase used to place desired gene in centre of (tetracycline) resistance gene
transform bacteria
grow on plate containing (ampicillin)
grow on plate containing (tetracycline)
any that grew on ONLY (ampicillin) and NOT (tetracycline) contain recombinant plasmids

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22
Q

what alternative gene markers do we use?

A

fluorescent green gene and test with UV light - any that don’t fluoresce contain recombinant DNA
gene producing lactase, lactase turns specific substrate blue - any that are unable to change colour in substrate contain recombinant DNA.

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23
Q

what is reverse transcriptase?

A

enzyme that synthesises a complimentary strand of DNA from mRNA

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24
Q

what is a plasmid?

A

a small ring-shaped DNA molecule found in the cytoplasm of bacteria, often used as a vector during genetic engineering

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25
Q

what is a marker gene?

A

a gene that is deliberately put into a cell along with a donor gene to make it possible to identify cells that have taken up the donor gene

26
Q

what is the process involved in transformation?

A

cells and vectors mixed with calcium ions
and given heat/electric shock
this makes the membrane more permeable so plasmids can enter the cell

27
Q

what are the stages in identification?

A

cut recombinant plasmid with (ampicillin resistance gene) using the same restriction enzymes that were used to isolate the gene
insert gene into plasmid
transform bacteria
transfer to agar plate containing (ampicillin)
any transformed DNA should be able to grow on ampicillin

28
Q

what are the problems with identification?

A

some plasmids can close up before gene of interest can be incorporated into the plasmid
some bacteria will have recombinant plasmid while others will just have a normal plasmid - as few as 1%

29
Q
A
30
Q

what is annealing?

A

joining of the primers to their complimentary bases

31
Q

what is DNA polymerase?

A

an enzyme which manufactures DNA by joining nucleotides.

32
Q

what is a primer?

A

a short sequence of nucleotides with a set of bases complimentary to those at one end of each of the two DNA fragments

33
Q

what is taq polymerase

A

heat stable enzyme that replicates DNA

34
Q

what is a thermocycler

A

a computer controlled machine that varies temperatures precisely over a period of time

35
Q

what is polymerase chain reaction?

A

in vitro gene cloning
method of copying DNA fragments
we can copy a small minute sample of DNA millions of times to allow accurate forensic examination

36
Q

what does in vitro mean?

A

in glass (test tube)

37
Q

what does “in vivo” mean?

A

in living (cells)

38
Q

which, out of in vivo + in vitro is the quicker process?

A

in vitro (PCR)

39
Q

what is DNA electrophoresis?

A

method of separating out negatively charged DNA fragments by applying a current
smaller fragments move quicker towards the positive anode

40
Q

what is genetic councelling?

A

communication process with patients that involves discussing the chance of inherited conditions, helping patients to make informed decisions about genetic testing and reproductive options, and providing support at a time that can be stressful

41
Q

what is genetic fingerprinting?

A

technique to determine the genetic identity of an organism eg. in forensics, paternity cases, diagnostics, breeding programmes in conservation. depends on an organisms genome containing repetitive, non coding introns, which have core sequences unique to the individual

42
Q

what is genetic screening?

A

checking for individuals in a family for mutant allele eg. sickle cell disease

43
Q

What is meant by recombinant DNA technology?

A

The transfer of DNA fragments from one organism to another.

44
Q

Why does recombinant DNA technology work?

A

Because the genetic code is universal, and therefore transcription and translation occur by the same mechanism and result in the same amino acid sequence across organisms.

45
Q

Summarise the process of using reverse transcriptase to produce DNA fragments.

A

mRNA complementary to the target gene is used as a template. It is mixed with free nucleotides which match up to their base pairs, and reverse transcriptase which forms the sugar-phosphate backbone, to create cDNA (complementary DNA)

46
Q

Summarise the process of using enzymes to produce DNA fragments.

A

Restriction endonucleases (RE) cut DNA at specific sequences. Different REs cut at different points but one RE will always cut at the same sequence. Therefore using particular REs allows you to cut out a certain gene of interest.

47
Q

In which two ways can we amplify DNA fragments?

A

PCR
using host cells

48
Q

Describe the reaction mixture in the first stage of PCR.

A

Contains the DNA fragment to be amplified, primers that are complementary to the start of the fragment, free nucleotides to match up to exposed bases, and DNA polymerase to create the new DNA.

49
Q

Summarise the process of amplifying DNA fragments using PCR.

A
  1. Heated to break apart the DNA strands.
  2. Cooled to allow primers to bind.
  3. Heated again to activate DNA polymerase and allow free nucleotides to join
  4. New DNA acts as a template for next cycle.
50
Q

Summarise the process of inserting a DNA fragment into a vector.

A

A plasmid (circular DNA from bacteria) is used as the vector, and is cut using the same restriction enzymes as the DNA, so that the ends are complementary. DNA ligase joins the fragment and plasmid together.

51
Q

Summarise the process of inserting a vector into a host cell.

A

Known as cell transformation. The host cells (bacteria) are mixed with the vectors in an ice-cold solution, then heat shocked to encourage the cells to take up the vectors. The cells can then be grown and the DNA fragment will be cloned.

52
Q

Summarise the process of identifying transformed cells.

A

Marker genes e.g. coding for fluorescence can also be inserted into vectors along with the DNA. When cells begin to grow, UV light can be used to identify which cells have taken up the vector and which haven’t.

53
Q

How can DNA probes be used to locate specific alleles?

A

The probe is designed so that its sequence is complementary to the allele you want to find.
They are labelled, amplified using PCR, then added to a sample of single stranded DNA.
The probe will bind if the allele is present.

54
Q

Give some applications of DNA probes.

A
  • screen someone’s DNA for a particular heritable health condition.
  • identify a gene for use in genetic engineering
  • predict how someone will respond to a drug.
55
Q

what is the purpose of DNA hybridisation?

A

To measure the degree of difference between two strands of DNA. Can be used to compare someone’s DNA to a certain gene to see if they have it.

56
Q

summarise the process of DNA hybridisation.

A

One DNA strand is labelled and mixed with an unlabelled comparison strand. The more similar the strands, the more strongly they will bind, and more energy will be required to break the strands apart.

57
Q

What are the benefits of genetic profiling?

A

Can identify heritable diseases very early, and therefore begin to treat them before symptoms develop, reducing impact on the individual.
Treatment can also be personalised to make it more effective.

58
Q

How does genetic fingerprinting work?

A

Every organism’s genome contains non-coding regions called variable number tandem repeats (VNTRs). The probability of two individuals having the same VNTRs is very low, so we can compare these areas to see if two DNA samples came from the same person.

59
Q

Summarise the process of genetic fingerprinting analysis.

A

DNA sample obtained, VNTRs cut out using restriction enzymes, labelled, and cloned using PCR. Fragments separated using gel electrophoresis. Banding patterns of each sample can then be compared.

60
Q

How does gel electrophoresis work?

A

DNA fragments are placed at one end of a slab of gel. An electric current is applied, causing the DNA fragments to move towards the other end of the gel. Shorter fragments travel further. The pattern of bands created is unique to every individual.

61
Q

Give applications of genetic fingerprinting.

A
  • Forensics
  • Medical diagnosis
  • Animal and plant breeding

Biology A2 content (19 decks)

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