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Gymnastics and tumbling > Chapter 25 ( Gene Technologies ) > Flashcards

Chapter 25 ( Gene Technologies ) Flashcards

1
Q

What is a PCR?

A

Polymerase chain reaction. Used to amplify the quantity of DNA. ( Generate many copies of the sample)
In vitro method carried out in thermocycler.

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2
Q

What does a PCR require?

A
  • DNA sample to be amplified
  • Excess of 4 nucleotide bases ( ACTG)
  • Small primer DNA sequences (usually 18-30 nucleotides long)
  • Taq DNA polymerase
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3
Q

What are primers needed in PCR?

A

Short sequences of DNA, complementary to one end of a fragment. They provide starting sequence for DNA polymerase to begin copying process.

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4
Q

What is Taq DNA polymerase?

A

Heat stable enzyme responsible for bonding the free nucleotide together to form copies of DNA fragments.
Obtained from thermophilic bacterium called Thermus aquaticus.

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5
Q

Sources of Contamination for PCR?

A
  • Equipment not thoroughly cleaned so DNA left on it
  • DNA from technician preparing sample, eg, dandruff.
  • Spores, bacteria, viruses in air.
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6
Q

Ways to reduce contamination during PCR?

A

Disposable equipment –> gloves, pipettes, head covers.

Sterile procedure.

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7
Q

Why is human DNA polymerase not used in PCR process?

A

Taq DNA polymerase is a heat stable enzyme that will not denature during PCR temperature cycling, whereas human DNA would.

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8
Q

Stages of DNA profiling?

A
  • Sample of DNA obtained
  • Sequences in DNA that vary between individuals selected and copied by PCR
  • Copied DNA split into fragments using restriction endonucleases.
  • Fragments separated by gel electrophoresis.
  • Band pattern compared to see if any are the same.
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9
Q

Uses of PCR?

A
  • DNA amplification of DNA extracted from fossils.
  • Forensic science, murder, using DNA samples from blood, semen, hair roots.
  • DNA profiling in paternity testing.
  • Detecting inherited diseases.
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10
Q

What is DNA profiling?

A

Procedure that can identify individuals based on their DNA.

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11
Q

What is electrophoresis?

A

Separates DNA fragments for identification and analysis based on their size.
Gel electrophoresis often performed after PCR has amplified DNA.

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12
Q

What are VNTRs?

A

Variable number tandem repeat.
Patterns of repeated nucleotides that are adjacent to each other in a DNA sequence.
eg, CAATT CAATT CAATT

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13
Q

Definition of genome?

A

Total genetic material of a living organism.

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14
Q

Why can VNTRs be used in genetic fingerprints in forensic science?

A

The probability of two unrelated people having same VNTR pattern is very low.

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15
Q

VNTR banding patterns info?

A

Gel electrophoresis can be used to produce VNTR banding patterns, which are compare during paternity testing. ( the child’s VNTR should match bands from either mum/dad )
Used in forensic crime investigations

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16
Q

What are SNPs?

A

Single Nucleotide Polymorphisms.
Sequences of DNA that vary between people by a single nucleotide
Occur in genes but are more common in non-coding DNA.
When occur in genes, called alleles
Causes by substitution mutations.

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17
Q

How are SNPs useful?

A
  • Can indicate a person’s susceptibility to disease and how they will respond to drugs, chemicals and vaccines.
    Drugs may produce different effects in individuals with different SNPs.

SNPs that are more common in people with a disease can be identified.

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18
Q

What DNA sequences are useful in genome studies?

A

VNTRs.
SNPs.
Haplotypes.

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19
Q

What are haplotypes?

A

Set of genes inherited together from one parent.

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20
Q

How are haplotypes useful?

A

Can provide insight into history of human migrations. As result of founder effect, people with same ancestral origins tend to share SNPs and very similar haplotypes.

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21
Q

Common haplotypes to analyse when studying ancestral origins?

A

Those in mitochondrial DNA and Y-chromosome DNA
Neither undergo recombination.
Mitochondrial DNA passes solely from mother to offspring.
Y-chromosome DNA passes solely from father to son

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22
Q

What is genetic engineering?

A

Joining together of DNA molecules from 2 different species that are inserted into a host organism to produce new genetic combinations.

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23
Q

Examples of genetic engineering?

A

Production of human insulin for diabetics. human growth hormone to treat dwarfism.

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24
Q

Definition of recombinant DNA

A

DNA that has been formed by joining together DNA from two different sources.

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25
Q

Definition of transformed organism

A

Organism that’s had its DNA modified

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26
Q

Definition of Transgenic organism

A

Organisms which have been genetically engineered to included a gene from a different species.

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27
Q

4 main steps of genetic engineering

A

Obtaining desired gene (Gene isolation)
Transduction (insertion of gene into vector)
Transformation into bacterial cells
Identifying transgenic bacteria

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28
Q

Definition of a vector?

A

A structure that carries a gene into a recipient cell during genetic engineering.
Eg, bacteriophages - viruses that infect bacteria.
Eg, plasmids - small circular molecules of DNA in bacteria.

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29
Q

What are reporter genes?

A

Used to identify bacterial cells that have taken up the new gene. Eg, some produce fluorescence that changes colour in a particular medium.

30
Q

What are restriction enzymes?

A

Endonucleases. DNA cutting enzymes that break the phosphodiester bonds within a DNA double helix at specific nitrogen-containing base sequences. Leave sticky ends.

31
Q

What are sticky ends?

A

The staggered cut produced by some restriction enzymes leaving single stranded sections. (exposed bases)
Base sequence of one strand is same as other strand read backwards. ( palindromic)
have complementary ends to sticky ends of other fragments.

32
Q

Name process in which two DNA fragments are sealed together

A

Ligation

33
Q

Definition of annealing?

A

When two matching sticky ends come together and join by base pairing.

34
Q

What have crops been genetically modified to do:

A

Be resistant to herbicides
Produce vitamin A (eg, golden rice)
Produce insecticides (eg, soya)
Combination of insecticide production and herbicide production.

35
Q

Uses of genetic engineering?

A

To produce human proteins in animals
To produce human proteins in crops
To produce genetically modified crops.

36
Q

Two ways of introducing DNA into plant cells?

A

Using a Ti plasmid

Using a gene gun

37
Q

What is a gene gun?

A

A device for delivering transgenes to cells.

38
Q

What are knockout mice?

A

Laboratory mice that are genetically engineered to have an inactivated gene, either because it was replaced or disrupted with an artificial piece of DNA.
The loss of gene activity causes changes in mouse’s phenotype, eg, change in appearance, behaviour, etc…

39
Q

What are knockout mice used for?

A

provide information about what the gene usually does.. Humans share many genes with mice so observing characteristics of knockout mice help to better understand how a similar gene may cause or contribute to diseases in humans.

40
Q

Examples of what knockout mice have been used to study?

A

Diabetes, heart disease, Parkinson’s disease, different kinds of cancer.

41
Q

Disadvantages of knockout mice?

A

Knocking out a gene may fail to produce an observable change in a mouse, or may produce different characteristics in humans when same gene is inactivated.

  • Some gene knockouts are lethal, so not all knockout mice develop into adults, which limits studies to embryonic development which makes it hard to determine a gene’s function in relation to human health.
42
Q

Why are Embryonic stem cells used when making knockout mice?

A

They are able to differentiate into nearly any type of adult cell, so when a gene is knocked out of ES cell, the effects can be observed in any tissue or adult mouse.

43
Q

What is gene therapy?

A

Specialised adaptation of genetic modification that allows genetic disorders to be treated.
Adds functional therapeutic alleles to cells with defective alleles within them. (defective alleles not removed)

44
Q

2 main problems with GT?

A

Getting functional genes into the defective cells

Making functional genes work once they are delivered into correct cells.

45
Q

What are the two types of gene therapy?

A

Somatic cell gene therapy

Germ line gene therapy

46
Q

What is SCID?

A

Severe combined immunodeficiency disease
Caused by faulty allele that codes for adenosine deaminase (ADA) ( globular enzyme)
Disease in which people don’t show a cell mediated immune response, so most die.
Virus was used to introduce normal ADA into T lymphocytes of patient using a retrovirus.
Some patients were diagnosed with leukaemia.
More recent SCID GT use stem cells from patient’s bone marrow.
therapeutic gene introduced into stem cells, injected back into patient.

47
Q

What is a retrovirus?

A

A virus that contains RNA and the enzyme reverse transcriptase to make a DNA copy of the RNA when they enter their host cell

48
Q

Methods for inserting therapeutic gene into target cells?

A

Vectors - eg, adenoviruses - cause respiratory diseases by inserting their DNA into lung cells so can be used to deliver functional alleles into lung cells that have faulty cystic fibrosis alleles.
Liposome - functional gene wrapped in lipid molecule. eg, bacterial plasmids that carry functional gene placed into liposomes. Liposomes sprayed into nostrils and inhaled. Liposomes cross phospholipid bilayer of csm and enter cells on lung.

Both ex vivo methods. - target cells removed from patient’s body , genes fixed, then put back into person.

49
Q

What are vectors?

A

methods to transfer the gene from one organism to another.

50
Q

What is cell mediated immunity?

A

Immune response that doesn’t involve antibodies.

51
Q

Cystic fibrosis treatment trials

A

insert FTG into viral DNA of adenovirus.
adenovirus delivered via nasal spray and inhaled.
virus delivered FTG and viral DNA to cells of gas exchange systems of CF patient.

52
Q

Difficulties with using viral vectors, (disadvantages of gene therapy)

A

Difficult to control where virus inserts the gene.
if the gene is inserted into gene that controls cell division it can cause cancers.
body could identify vectors as foreign - immune response.

53
Q

What is germ line GT?

A

Adds FTG in to a fertilised ovum

ensures all cells of organism will contain FTG, so FTG can be passed on to next generation through gametes.

54
Q

Why are stem cells ideal for use as host cells for GT?

A

stem cells are unspecialised
only need to insert therapeutic gene into one cell
they are pluripotent
the modified stem cells will develop into any tissue required.
No immune response triggered in patient’s own cells used.

55
Q

Differences between somatic GT and germ line GT?

A

Somatic GT: effects often short lived, confined to individual only, few ethical issues.
Germ line GT: results in permanent changes passed down to subsequent generations, effects individual and offspring, many ethical issues.

56
Q

Ethical implications of gene therapy in disease treatment?

A

Has potential for unforeseen negative effects on future generations
New genes may promote immune response.
Some view it as unnatural. Should we alter genome of unborn children. - abuse of principle.

57
Q

Advantages of gene therapy?

A

Prolong lives of those with life threatening issues, and improves quality of life.
Germline therapy allows carriers of genetic disorders to conceive baby without genetic disorders.

58
Q

What are exons?

A

Coding regions of a gene

59
Q

What are introns?

A

Non coding regions

60
Q

What is RNA splicing?

A

mRNA formed immediately after transcription called pre-mRNA with both introns and exons.
introns removed
exons spliced together
Then mature mRNA translated to produce ppc.

61
Q

Advantage of exon splicing?

A

enables different mRNA molecules to be formed from 1 gene.

62
Q

What is alternative splicing?

A

Some introns retained within mature mRNA and exons joined in different combinations.
ADV - 1 gene can code for many different proteins, so organisms can carry fewer genes in genome.

63
Q

What is RNA interference? (RNAi)

A

sections of DNA produce short RNA strands that bind to mRNA, effectively blocking protein synthesis. Gene expression is inhibited, so genes are ‘silenced’

64
Q

Where does RNAi occur?

A

In animal cells, plant cells, fungal cells, and some bacteria.

65
Q

2 forms of RNAi?

A

small interference RNA ( siRNA) + microinterference RNA (miRNA)

66
Q

Enzymes used in genetic engineering?

A

Restriction enzymes
DNA ligase
(DNA polymerase)
(reverse transcriptase)

67
Q

what transgenic organisms can produce human proteins?

A

Cows - contains human gene for lysozyme (antibacterial properties)
Goats - contains human gene for antithrombin (prevents blood clotting)
tobacco - contains human gene for antibodies (protection from pathogens)
rice - contains human gene for human serum albumin (blood transfusions)

68
Q

Advantages of using transgenic plants to produce human proteins?

A
  • Low production costs
  • Large scale processing infrastructure already in place for most crops
  • scale-up is rapid and efficient only requiring cultivation of additional land.
69
Q

What are RNAi?

A

Used to silence genes ( switch genes off)
mRNA existence is short-lived, so degrading it - reduction in protein synthesis.

Either by destroying mRNA or preventing ribosomes translating mRNA.

70
Q

Types of iRNA?

A

siRNA (small interference RNA)

miRNA ( microRNA)

71
Q

Steps in iRNA.

A
  • Double stranded precursors of miRNA and siRNA bind to a dicer. (endonuclease protein)
  • Dicer cuts precursor into short segments
  • Short double stranded RNA binds to Argonaute protein.
  • 2 strands unwind, and one of them stays bound to protein (guide strand)
  • iRNA and argonaute protein = RNA-induced silencing complex. (RISC)
  • This complex binds to specific mRNA by complementary base pairing.
  • Binding causes RISC to cut mRNA molecule in two. ( or makes mRNA not able to fit into ribosome so no translation)
  • Cleaved mRNA no longer used in translation, so broken down by nuclease enzymes.
72
Q

Applications of iRNA?

A

Identifying function of genes by silencing gene and observing functional effects.

Genetically modifying crop plants by silencing undesirable genes, eg, those that make toxins or allergens.

Treating human patients to silence essential genes in cancer cells.

Gymnastics and tumbling (29 decks)

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