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Biology Unit 5 > DNA Technology > Flashcards

DNA Technology Flashcards

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1
Q

Name the stages involved in the process of making a protein using DNA technology.

A

Isolation - of DNA fragments with the gene for a desired protein
Insertion - of the DNA fragment into a vector
Transformation - transfer the DNA to a host cell
Identification - using gene markers
Growth/cloning - the host cells

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2
Q

What is recombinant DNA?

A

The DNA of two different organisms that had been combined

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3
Q

The use of reverse transcriptase

A

An enzyme that catalyses the production of DNA FROM RNA

Used to change the RNA from the retroviruses into DNA

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4
Q
  1. Isolation using reverse transcriptase
A

A cell that readily produces the protein is selected.
They have large quantities of the relevant mRNA
Reverse transcriptase is used to make DNA from RNA - cDNA (complimentary DNA)
DNA polymerase builds up the complimentary nucleotides on the cDNA template

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5
Q
  1. Isolation using restriction endonuclease
A

Enzymes that cut up viral DNA
Each one cuts a DNA strand at a specific sequence of bases - recognition sequence
Sometimes cut at two opposite base pairs (blunt ends)
Or a staggered fashion exposing unpaired bases (sticky ends)

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6
Q

What does ‘in vivo’ mean?

A

The DNA fragments are cloned by transferred to a host cell using a vector

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7
Q

What does ‘in vitro’ mean?

A

The DNA fragments are cloned using the polymerase chain reaction

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8
Q

How is recombinant DNA formed?

A

The restriction endonuclease cuts out a DNA fragment, leaving a sticky end
DNA from another source cut with the same endonuclease joins the other section using DNA ligase

DNA ligase joins the phosphate-sugar framework

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9
Q

What is a vector?

A

A carrying unit used to transport the DNA into the host cell

- usually a plasmid

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10
Q
  1. Insertion using restriction endonuclease
A

Restriction endonuclease breaks the plasmid loop at an antibiotic resistance gene
(The same restriction endonuclease used to cut out the DNA fragment)
DNA fragments are mixed with the opened up plasmid, becoming incorporated
Joined by DNA ligase
Recombinant DNA is formed

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11
Q
  1. Transformation
A

The plasmids are then reintroduced into bacterial cells.
Bacteria cells and the plasmids are mixed in a calcium ion medium, making the bacteria permeable for the plasmid to enter

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12
Q

Why do not all the bacterial cells possess the DNA fragments?

A

Only a few bacterial cells take up the plasmids when they are mixed

Some plasmids do not incorporate the DNA fragment

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13
Q

How are the antibiotic resistance genes used to identify which bacterial cells have taken up the plasmid?

A

Resistance to ampicillin and tetracycline

  • All bacterial cells are grown in a medium containing ampicillin
  • Bacteria cells that have taken up the plasmid have resistance to it
  • The cells therefore survive
  • The cells that don’t take it up aren’t resistant and die
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14
Q
  1. Identification using gene markers
A

Gene markers are:
Resistant to an antibiotic
Make a fluorescent protein
Produce an enzyme with identifiable actions

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15
Q

Fluorescent markers

A

A gene from a jellyfish is transferred into the plasmid and produces a green fluorescent protein (GFP)
Those that have taken up the plasmid with the gene that is to be cloned will NOT produce GFP

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16
Q

Enzyme markers

A

A gene that produces lactase as this turns a particular colourless substrate blue
If a plasmid with the required gene is present it will NOT produce lactase

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17
Q

Antibiotic resistance markers

A

Replica plating - a technique to identify the cells with the plasmids that have taken up the new gene

This uses the other resistant gene in the plasmid - the one cut to incorporate the required gene
Gene for resistance to tetracycline is but so will no longer produce the enzyme to break down tetracycline

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18
Q

Replica plating

A

The bacterial cells that survive the ampicillin have taken up the plasmid - these are cultures on an agar plate
A sample of each colony is transferred onto a replica plate
The replica plate contains tetracycline
Colonies that die took up the required gene
These can then be identified on the original plate

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19
Q

PCR - what does this process require?

A 
The DNA fragment to be copied 
DNA polymerase to join nucleotides 
Primers 
Nucleotides
Thermocycler - a computer-controlled machine that varies the temperature precisely
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20
Q

What are primers?

A

Short sequences of nucleotides with complementary base pairs to those at one end of each of the two DNA fragments

21
Q

First stage of PCR

A

Separation of DNA stands
DNA fragments, primers and DNA polymerase are placed in the termocycler
Temperature increased to 95C causing the strands to separate

22
Q

Second stage of PCR

A

Addition (annealing) of primers
Mixture is cooled to 55C
Primers join to their complimentary bases on the DNA fragment
Provide the start sequence for DNA polymerase and prevents the two strands from rejoining

23
Q

Third stage of PCR

A

Synthesis of DNA
Temperature is increased to 72C - optimum temperature for DNA polymerase
Adds complimentary nucleotides along the DNA strands, beginning at the primer until it reaches the end of the chain

24
Q

Advantages of in vivo

A

Useful when introducing a gene into another organism
Almost no risk of contamination
Very accurate
Cuts out a specific gene so very precise
Can be produced in large quantities

25
Q

Advantages of in vitro

A

Extremely rapid - in a matter of hours 100 billion copies can be made
Doesn’t require living cells

26
Q

Use of recombinant DNA

A 
Genetic modification - when the genetic make up of organisms are altered by transferring genes they can:
Increase yield of animals or crops
Increase nutrient content of food 
Introduce resistance to disease or pests
Make plants tolerant to herbicides
Develop tolerance to extreme weather
27
Q

Genetically modified microorganisms

A

Antibiotics - naturally produced by bacteria GM can increase quantity of antibiotics produced

Hormones- insulin can be incorporated into bacterial cells with no effects to the patient

Enzymes - manufactured by modified bacteria

28
Q

What causes cystic fibrosis?

A

A mutant recessive allele where there’s a deletion mutation
CFTR gene produces a non functional protein
CFTR is a chloride- ion channel protein

29
Q

What happens in a person with cystic fibrosis?

A

The non functional channel protein prevents chloride ions from leaving the cell
Water is retained in the cell as the concentration gradient causes osmosis into the cell

Causes breathing difficulties
Thick mucus in the pancreatic ducts and sperm ducts
Mucus congestion leading to risk of infection

30
Q

Treating cystic fibrosis using gene therapy: gene replacement

A

The defective gene is replaced with a healthy gene.
Germ-line gene therapy - replacing the gene in the fertilised egg
All cells will develop normally and be passed on to their offspring
Permanent solution affects future generations
Process is illegal due to ethical issues

31
Q

Treating cystic fibrosis using gene therapy: gene supplementation

A

Copies of the healthy gene are added alongside the defective, masking the defective gene
Somatic-cell gene therapy - targets affected tissues
Not passed onto offspring
The cells continually die and are replaced so treatment is needed periodically- limited success
Long term is to target undifferentiated stem cells therefore lasting their lifespan

32
Q

How is the cloned CFTR genes delivered in somatic-cell gene therapy?
A harmless virus

A

Adenovirus causes colds by injecting their DNA into the epithelial cells of the lungs
Useful vectors to deliver this gene then
In vivo gene cloning is used
The adenoviruses are introduced it or he nostrils of the patient

33
Q

How is the cloned CFTR genes delivered in somatic-cell gene therapy?
Lipid molecules

A

Genes are wrapped in lipid molecules as these can easily pass through the phospholipid bilayer
In vivo gene cloning is used
The plasmids are removed from the bacteria and wrapped in lipid molecules forming liposomes
These are sprayed into the nostrils of the patient and drawn down into the lungs

34
Q

Problems with how CFTR is delivered in gene therapy

A

Adenoviruses may cause infections
Patients may develop immunity to the adenoviruses
Liposomes may be too large to pass through
May not be expressed

35
Q

What is Severe combined immunodeficiency (SCID)?

A

A rare inherited disorder where the sufferer doesn’t show a cell mediated immune response or produce antibodies
Arises when the individual inherits a defect in the gene that codes for the enzyme ADA
ADA destroys toxins that would kill WBC

36
Q

How is SCID treated?

A

ADA from a healthy human is inserted into a retrovirus
They’re mixed with the patients T cells
ADA is inserted into the T cells
T cells are reintroduced into the patients blood

Success is limited because T cells only live for 6-12 months so has to be repeated

37
Q

What is a DNA probe?

A

A short, single-stranded section of DNA that has a label attached that makes it easily identifiable
Two common ones used are:
Radioactively labelled probes
Fluorescently labelled probes

38
Q

How are DNA probes used?

A

Has complimentary bases to a portion of DNA that makes up part of the gene whose position we want to find
DNA being tested has been treated to separate its two strands
Separated DNA strands are mixed with the probe which binds to complimentary bases on the strand - DNA hybridisation
Site at which it binds can be identified

39
Q

DNA sequencing

What is the Sanger method?

A

Uses modified nucleotides that cannot attach to the next base sequence when they are being joined together
They act as terminators, ending synthesis of a DNA strand

40
Q

What is in the four test tubes set up for the Sanger method?

A

Single stranded DNA fragments which act as a template for the synthesis of it’s complimentary strand
Mixture of nucleotides
Small quantity of one of the 4 terminator nucleotides (tube 1 - Adenine etc)
A primer to start DNA synthesis (labelled)
DNA polymerase to catalyse DNA synthesis

41
Q

Why are there different lengths of DNA fragments in DNA sequencing?

A

Binding of nucleotides is a random process so the chance of either a terminator or normal nucleotide binding is equally likely
DNA synthesis may be terminated after a few nucleotides or after a long fragment, depending on where the terminator binds

42
Q

How can the section of DNA be identified in DNA sequencing?

A

DNA fragments will all end in the same base (tube 1 - Adenine etc)
They can be identified because the primer attached to the other end of the DNA section is labelled

43
Q

What is the stage in which the different lengths of DNA fragments are separated out?

A

Gel electrophoresis

44
Q

What happens in gel electrophoresis?

A

The DNA fragments are placed on an agar gel
A voltage is applied across it
Larger fragments move slower so smaller ones move further
A sheet of photographic film is placed over the agar gel
Radioactivity from each fragments exposes the film showing where it is situated on the gel

45
Q

DNA fragments sequenced have to be less than around 500 bases.

A

Larger genes and whole genomes must therefore be cut into smaller fragments by RESTRICTION ENDONUCLEASE

46
Q

What does restriction mapping involve?

A

Involves cutting DNA with a series of different restriction endonucleases.

47
Q

How are the fragments separated in restriction mapping?

A

By electrophoresis

48
Q

How is DNA sequencing and restriction mapping carried out by machines?

A

A fluorescent dye is used as a label the DNA primer instead of a radioactive labels
Each base takes up a different colour
Electrophoresis is carried out and results are scanned by lasers to show what the bases are

Biology Unit 5 (8 decks)

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