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Manipulating genomes Flashcards

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1
Q

what is the role of a PCR test on DNA?

A

amplifying sections of DNA to be analysed
produce large quantities of a specific target sequence of DNA

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2
Q

part of the PCR test is to heat the DNA to 90-95 degrees , why is this done?

A

break the hydrogen bonds between bases of the two strands so they can be separate

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3
Q

what is the role of primers within the PCR test?

A

two primers designed to select the sequence of DNA you want to copy
done through complementary base pairing

also act as a starting point for Taq Polymerase

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4
Q

when working with two primers for base pairing , where does each primer attach?

A

one primer attaches to top strand at one end of the sequence

other primer attaches to bottom strand at the other end

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5
Q

Explain why the enzyme Taq polymerase , found in hot springs, is used instead of eukaryotic DNA polymerase?

A

Taq’s optimum temperature = 72C , much higher than most organisms so able to function at high temperatures used in the PCR without denaturing

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6
Q

what is a genome?

A

all genetic material in an organism

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7
Q

describe the steps taken for a PCR test , there’s 6 steps

A
  1. for DNA to be amplified needs to be mixed with primers, Taq polymerase, good supply of the four nucleotides and a suitable buffer, then put into PCR machine
  2. DNA sample needs to be heated to 90-95 degrees for 30 seconds to separate the two strands
  3. then sample cooled down to 50-55 degrees to allow the primers to anneal (bind) to the DNA strand
  4. sample then heated to Taq polymerase optimum temperature (72 degrees)
  5. once primers binded to dna template strand , taq polymerase attaches to the primers to add nucleotides
  6. each reaction then doubles the amount of DNA
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8
Q

what is agarose gel electrophoresis?

A

the separation of DNA fragments based on size

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9
Q

Is DNA positive or negative?

A

negative

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10
Q

what is mini-satellite within electrophoresis?

A

10-100 base sequence
repeated 50 to several hundred times

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11
Q

whats micro satellite in electrophoresis?

A

2-6 base sequence
5 to 100 times

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12
Q

how does electrophoresis demonstrate close evolutionary relationship?

A

similar gene sequence
similar proteins have similar binding patterns on the gel

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13
Q

How is DNA separated in electrophoresis?
what orders it

A

size of fragments

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14
Q

what are the charges of a cathode and anode?

A

cathode = negative
anode = positive

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15
Q

why is DNA stained in electrophoresis?

A

to show contrast

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16
Q

what is the role of agarose gel in electrophoresis?

A

provides resistance to follow of DNA

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17
Q

Will DNA move towards the positive or negative electrode in electrophoresis?

A

positive bc dna negative

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18
Q

describe the steps of electrophoresis?

A
  1. restriction enzyme used to cleave DNA into smaller segments to produce various sizes
  2. DNA segments loaded into wells in a porous gel
    - the gel will float in a buffer solution
    - chamber between two electrodes
  3. current passes through chamber which causes the DNA fragments to move toward positively charged cathode
  4. smaller fragamets move faster and further than larger
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19
Q

in electrophoresis what size of dna fragments move faster?

A

smaller

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20
Q

what are the types of vectors used in genetic engineering?

A

viruses
plasmids
naked DNA
liposomes

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21
Q

what is an advantage of using a viral vector for somatic gene therapy?

A

can enter any cell in the body
cheap
delivers desired gene

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22
Q

what is a disadvantage of using a viral vector for somatic gene therapy?

A

cancer
unknown affect

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23
Q

which is permanent
somatic or germline cell therapy?

A

Germ line

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24
Q

what does somatic mean?

A

Body

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25
does germ line theory target a specific tissue or every future tissue?
Every future tissue
26
Can somatic cell gene therapy outcome be passed onto offspring? Yay or Nay?
NAYYY It cannot
27
does the affect of germ line therapy be passed onto offspring?
Yes its passed onto offspring
28
out of somatic or germ line cell therapy, which one has the gene introduced into the body/non-reproductive cell/ embryo?
somatic grem line in sperm/ egg
29
does the affect on somatic cell therapy long term or short term?
Short term
30
is the affect on germ line therapy long or short term?
Long term
31
out of somatic and germ line therapy, which one affects some cells and which one affects all cells?
somatic = some germ line = all cells get functional gene
32
what is a real life example of gene therapy? describe briefly the process?
human insulin production in human cell insulin gene removed in bacterial cell plasmid remove insulin gene and plasmid combined placed into a transgenic bacteria Then into fermenter
33
what does in vitro mean?
new gene inserted into cells inside the body via vector
34
what does ex vivo mean?
new gene inserted into cells outside body via virus vector
35
define gene therapy?
using various mechanisms to alter a persons genetic material / cure diseases
36
what are two types of gene therapy?
Germ line Somatic cell gene therapy
37
for somatic cell gene therapy to happen/ work what does it require?
a vector
38
what are the examples of vectors needed for somatic cell gene therapy?
viruses non-viral vectors
39
viruses are vector used for somatic cell gene therapy, name the 3 types of viruses and roles?
1. retroviruses + lentiviruses = inserted into host DNA = modified not to divide = can cause cancer 2. Adenoviruses = not inserted into host cell = doesn't pass gene onto future cells
40
name the two types of non- viral vectors used in somatic cell gene therapy?
liposomes naked DNA
41
is germ line cell therapy permanent Y/N? and reversible? Y/N?
permanent = yes reversible = no , irreversible
42
describe an example of germ line cell therapy when have a fault mitochondria
patient couple with faulty mitochondria donar couple with healthy mitochondria 1. healthy nuclear DNA removed from faulty mitochondria 2. nucleus removed from healthy mitochondria 3. healthy mitochondria combined with healthy nuclear DNA
43
is germ line cell therapy legal in humans?
no because its permanent and can be inherited by future generation
44
What is attached to the ddNTP containing the terminator base?
Radioactive tag
45
What is different about the sugar in a ddNTP during gene sequencing?
No hydroxyl group on the 3’ end
46
Once gene sequencing has taken place via sangers method, how are the DNA fragments analysed?
By autoradiography
47
since sangers method of gene sequencing , this has been improved by technology How? What has changed?
1. Many strands of DNA sequenced at once 2. Use lasers to visualise the ddNTP’s which speeds up data processing 3. Doesn’t involve electrophoresis 4. Most methods of sequencing now automated rather than manually
48
Why would high throughput sequencing (sangers method being improved by technology) be useful? What can it be used for? 3 things
1. Evolutionary biology and classification 2. Personalised medicine 3. Disease diagnosis
49
define DNA sequencing? (1)
working out the sequence of bases / nucleotides
50
state three ways gene sequencing is used?
1. comparisons between individual species and between species 2. predict sequences of amino acids in polypeptides 3. used in the development of synthetic biology
51
role of terminator base in Sangers method?
stop DNA replication
52
53
Describe the steps to Sangers gene sequencing method
1. Densature double strand to separate the strands 2. Per used to replicate strands to produce large amount of copies to be analysed 3. Copies added to 4 reaction vessels and DNA polymerase added 4. Nucleotides modified by terminator bases (ddNTPs) and are labelled with radioactive tag which one in each vessel 5. Terminator base stops DNA replication at random nucleotide base sites , which creates DNA fragments to be different sizes 6. Fragments of DNA separate by electrophoresis and analysed by autoradiography
54
What does bioinformatics allow to happen?
Rapid comparison of sequences via computational analysis
55
Within bioinformatics what does the database contain information off?
DNA sequencing mRNA sequences Protein structure
56
Why is bioinformatics helpful?
1. Compares evolutionary relationships 2. Looks at diseases 3. Review genotypes and phenotypes
57
What is synthetic biology and why is it used?
Man made biology where nature redesigned Cheaper than altering bacteria
58
What is genetic modification also known as?
Genetic engineering
59
Genetic modification can happen to what organisms?
Bacteria Plants Animals
60
Why can genetic modification take genes from one species and insert them into the genome of another species without worrying about the evolutionary relationship?
Genetic code is universal All living things use the same bases and sequence of amino acids Same codons code for the same amino acids in all organisms
61
What is the role of a restriction enzyme within genetic modification?
Cut out the desired gene from a genome and cut out the plasmid from a bacterial cell like e.coli
62
What are ‘sticky ends’ in genetic modification?
Same bases that have been left exposed once gene removed
63
Describe the process of genetic modification The steps involved!
1. Cut the desired gene from a genome by using a restriction enzyme 2. E.coli bacteria contains plasmids which are removed by the Same restriction enzyme 3. Causes sticky ends on both the genome and plasmid 5. Ligaments joins the sticky ends together and fixes the gene and plasmid together 6. Recombinant plasmid inserted into host cell and new gene expressed
64
Why is the same restriction enzyme used within gene modification?
So the ‘sticky ends’ exposed from the genome and plasmid are the same for them to be joined together
65
What vectors does genetic modification use?
Plasmids and vectors
66
What are some helpful characteristics that would be looked for , for a GMO crop?
Frost resistance Disease resistance Longer shelf life Higher yield
67
Name some risks of genetically modified crops?
Toxins can cause allergic reactions in humans Transferred gene could mutate Increase resistance to pests Biodiversity reduced

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