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Biology - Module 6 > Manipulating genomes > Flashcards

Manipulating genomes Flashcards

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1
Q

what is DNA sequencing

A

allows for the nucleotide base sequence of an organism’s genetic material to be identified and recorded

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2
Q

methods of DNA sequencing

A

sanger method
high-throughput sequencing

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3
Q

what is the sanger method also known as

A

chain termination method

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4
Q

overall what is the sanger method

A
  • Dideoxynucleotides pair with nucleotides on template strand in DNA replication
  • When DNA polymerase encounters dideoxynucleotide on developing strand – stops replicating
  • Termination method
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5
Q

describe the sanger method

A

4 test tubes – each with diff type of dideoxynucleotides ( A* / T* / C* / G*)

Test tubes incubated – temp allows enzyme function

96 degrees - break H bonds between DNA

50 - primer anneals

60 - DNA polymerase

Primer anneals to start of single stranded template – short section of double stranded

DNA polymerase attached to double stranded section + begins DNA replication using free nucleotides in test tube

At any time – DNA polymerase inserts on dideoxynucleotide by chance – results in termination of replication

Complementary DNA chains varying lengths made

New complementary DNA separated from template DNA

Resulting single stranded DNA separated according to length via gel electrophoresis

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6
Q

what is a primer

A

short single-stranded sequence with set of bases complementary to those at the start of the DNA fragment

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7
Q

what needs to be in each test tube for the sanger method

A

DNA to be sequenced as single-stranded template

DNA polymerase

DNA primers

free nucleotides

one of 4 types of dideoxynucleotides ( A* / T* / C* / G*)

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8
Q

when separated how do you actually know the sequence from sanger method

A

Each test tube only has 1 type of dideoxynucleotide – so you can know terminal nucleotide of each fragment

unique position on electropheresis gel because of unique mass

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9
Q

how would electropheresis separate these fragments

A

gel will have four wells, one each for A, C, T, and G

fragment that consists of only one nucleotide will travel all the way to the bottom of the gel, and every band above this on the gel represents the addition of one more base

allows the base sequence to be built up one base at a time

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10
Q

overall high-throughput sequencing

A

New methods of sequencing DNA that are automated, very rapid and cheaper than orig. methods

uses capillary electropheresis

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11
Q

Capillary gel electrophoresis

A

Separates macromolecules such as nucleic acids through capillary action in a capillary tube

high resolution

capable of separating chains of DNA that vary by only one nucleotide in length

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12
Q

capillary gel electropheresis method

A

each type of dideoxynucleotide labelled using fluorescent dye

 adenine – green
 thymine – red
 cytosine – blue
 guanine – yellow

laser beam used to illuminate all dideoxynucleotides

detector reads colour + position

feeds into computer

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13
Q

bioinformatics

A

storage, retrieval, and analysis of data from biological studies

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14
Q

computational biology

A

using computers to study biology – create simulations / models

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15
Q

important of genome sequencing

A

allows to make comparisons with genomes of other organisms – human genome project

  • find degree of similarity = how closely related they are
  • useful for looking at organisms that can be used as a model for humans
  • evolutionary relationships

allows us to understand genotype-phenotype relationships

  • target specific base sequences to knock out + observe effect

aid research + disease control

  • genomes of pathogens can be sequences + analysed
  • highly infections strains are identified
  • personalised medicine
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16
Q

proteome

A

full range of proteins produced by genome

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17
Q

why is the proteome difficult to determine

A

non-coding DNA + regulatory genes + alternative splicing

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18
Q

proteome or genome larger

A

proteome is larger than the genome due to:

Alternative splicing

Post-translational modification of proteins (often takes place in the Golgi apparatus)

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19
Q

Synthetic biology

A

aims to create new biological parts, devices, and systems, or to redesign systems that already exist in nature

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20
Q

what does synthetic biology involve

A

involves large alterations to an organism’s genome

operate in novel way more than genetic engineering

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21
Q

Computational bio

A

Uses data from bioinformatics to build theoretical models of biological systems which can be used to predict what happens in diff circumstances

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22
Q

How can bioinformatics help determine whether a newly sequenced allele causes genetic disease

A

Base sequence of normal allele and known alternatives held in database as well as AA sequence

Computational analysis allows rapid comparison of sequences w/ newly sequenced alleles

Can create model of new protein structure

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23
Q

Uses of computational bio

A

Analysing base pair in DNA

Working out 3D structures of proteins

Understanding molecular pathways e.g. gen reg

Identify genes linked to spp diseases

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24
Q

Benefits of using DNA sequencing in studying epidemiology of infectious disease

A

Allows you to identify pathogen

Sequence DNA and compare to sim microorganisms

Faster than trad methods e.g.culturing bacteria

Can follow routes of infection

Cn identify carriers

Can help find drugs

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25
Q

Variable number tandem repeats

A

Short nucleotide sequence that is repeated throughout the genome

the number of this varies at any given locus in the genome

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26
Q

how is the number of VNTR repeats determined

A

inherited

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27
Q

what would a high similarity of VNTRs indicate

A

closely related

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28
Q

describe the process of genetic profiling / fingerprinting

A

Obtain the DNA = extracted from the root of a hair / spot of blood / semen / saliva

Increase the quantity of DNA by using PCR

Use restriction endonucleases to cut the amplified DNA molecules into fragments

Separate the fragments using gel electrophoresis

gel immersed in alkali - separate DNA double strands into single strands

transferred onto a membrane by southern blotting

Add radioactive or fluorescent probes in excess

complementary + bind to specific VNTR regions - hybridisation

X-ray images are produced or UV light is used to produce images of the fluorescent labels glowing.

These images contain patterns of bars (the DNA profile) which are then analysed

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29
Q

how do the restriction endonucleases work in DNA profiling

A

different restriction endonucleases - cut at different recognition / restriction sites

make two cutes - once through each strand of DNA double helix

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30
Q

hybridisation

A

radioactive / fluorescent DNA probes added in excess to single stranded DNA fragments on the membrane

bind to complementary strands of DNA

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31
Q

southern blotting

A

DNA (-ve) from gel electrophoresis is transfereed to a +vely charged membrane e.g. nylon

Fragments are irreversibly bound to the blot, whilst maintaining their relative positions on the gel

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32
Q

DNA probes

A

Single stranded short piece of DNA with a known complementary sequence to the VNTR
Synthesised chemically and is radio-labelled

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33
Q

how would you see the result of DNA profile if you added radioactive labels to the DNA probes

A

X ray images of the membrane

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34
Q

how would you see the result of DNA profile if you added flourescent labels to the DNA probes

A

membrane placed under UV light so fluorescent tags glow

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35
Q

uses of DNA profiling

A

Tissue typing

= Donor + recipient tissues matches to reduce risk of rejection

Detection of oncogenes

= Can inform medication

Detect mutations

= E.g. – embryo selection

Identify type of viral infection + monitor spread of infectious disease

= PCR covid testing

Identify suspects of crimes // forensic science

= DNA profile of sample compared to samples from suspect / criminal database / victim

= Identify bodies

determine familial relationships for paternity cases

species conservation to help scientists with captive breeding programmes to reduce chances of inbreeding

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36
Q

PCR

A

polymerase chain reaction

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37
Q

what is PCR used for

A

in vitro method of DNA amplification

used to produce large quantities of specific fragments of DNA/RNA from very small samples

even just 1 molecule of DNA / RNA

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38
Q

PCR ingredients

A

small sample of target DNA

2 primers

Taq polymerase

Free nucleotides

Buffer

Thermal cycler

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39
Q

what are the primers complementary to in PCR

A

Complementary to the 3’ end

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40
Q

why do you need 2 primers for PCR

A

One for each single strand of the now broken double helix

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41
Q

what is Taq polymerase + where is it found

A

DNA polymerase

from thermophilic bacteria in hot springs

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42
Q

why do we use taq polymerase

A

does not denature at high temp

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43
Q

why do we need a buffer in PCR

A

optimum pH for reactions beaker

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44
Q

why do we need a thermal cycler

A

automated

provided correct temp for correct time

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45
Q

3 stages of PCR

A

denaturation

annealing

elongation / extension

46
Q

describe PCR - denaturation

A

DNA fragments / primers / DNA polymerase / nucleotides added to thermocycler

heated to 95°C

breaks the hydrogen bonds

forms 2 separate strands with exposed nucleotides

denaturation – separating strands

47
Q

describe PCR - annealing

A

temperature is decreased to between 50 - 60°C

primers (forward and reverse ones) can anneal to the ends of the single strands of DNA

48
Q

describe PCR - elongation

A

temperature is increased to 72°C for at least a minute

optimum temperature for Taq polymerase to build the complementary strands of DNA

produce the new identical double-stranded DNA molecules

49
Q

formula for total number of strands formed from PCR

A

Number of original DNA strands X 2^number of PCR cycles = total number of strands

50
Q

advantages of PCR

A

very rapid – millions of copies made in hours

does not require living cells – only base sequence

PCR – needed for the sanger method + DNA sequencing

51
Q

electropheresis

A

molecules are separated according to their size/mass and their net charge

52
Q

why does separation occur in electropheresis

A

electrical charge

mass / size

type of gel

53
Q

how does electrical charge aid separation

A

negatively charged DNA - negatively charged due to the phosphate groups

when placed in an electric field the molecules move towards the anode

54
Q

how does size aid separation

A

Different sized molecules move through the gel at different rates

tiny pores in the gel result in smaller molecules moving quickly + larger molecules slowly

55
Q

how does type of gel aid separation

A

Different gels have different sized pores

affect the speed at which the molecules can move through them

56
Q

process of electropheresis for DNA

A

Create an agarose gel plate in a tank

Wells are cut into the gel at one end

Submerge the gel in buffer solution

Load the fragments into the wells using a micropipette

Apply an electrical current to the tank.

The smaller mass / shorter pieces of DNA fragments will move faster + further from the wells

smallest fragments reach end - turn off electric current

gel placed in alkaline buffer solution

The fragments are not visible

must be transferred onto absorbent paper or membrane via southern blotting

fixed in place by UV / heat

Probes are then added

X-ray image is taken or UV-light is shone onto the paper producing a pattern of bands which is generally compared to a control fragment of DNA

57
Q

why do we put the final gel in a alkaline buffer solution

A

denature DNA fragments

two strands separate

58
Q

why are probes added in electropheresis

A

allow VNTRs / sequences to be identified

A radioactive label which causes the probes to emit radiation that makes the X-ray film go dark, creating a pattern of dark bands

A fluorescent stain / dye which fluoresces (shines) when exposed to ultraviolet (UV) light, creating a pattern of coloured bands

59
Q

how are proteins prepared for electropheresis

A

Denaturing (to break the disulfide bonds)

manipulating the proteins into rod shapes (negatively charged) to allow separation by size

60
Q

membrane for southern blotting

A

nylon membrane

61
Q

sanger method final product

A

DNA sequence of COMPLEMENTARY STRAND

62
Q

overall steps of genetic enginnering

A

isolate the gene

multiply DNA fragment - PCR

formation of recombinant DNA

transferring the vector

63
Q

what are the two ways to isolate a gene

A

cut the DNA or isolate mRNA

64
Q

describe isolating gene - cutting DNA

A

restriction endonucleases cut 2 DNA fragments unevenly

sticky ends – regions with unpaired / exposed bases

make it easier to insert the desired gene into DNA of diff organism

65
Q

difference in cuts in DNA profiling + genetic enginnering

A

blunt cuts vs sticky ends

66
Q

describe isolating gene - mRNA

A

isolating mRNA for gene – reverse endonucleases

using reverse transcriptase

produce single strand of complementary DNA
- cDNA

67
Q

advantages of isolating mRNA

A

easier to identify desired gene – particular cell will make very specific types of mRNA +

mRNA + cDNA – no introns

e.g. – beta cells in pancreas make insulin – a lot of insulin mRNA

68
Q

what do you do after you isolate the gene

A

multiply DNA fragment - PCR

69
Q

what do you do after you multiply DNA fragment - PCR

A

form recombinant DNA

70
Q

how do you form recombinant DNA

A

Same restriction endonuclease cuts plasmid

Complementary sticky ends to DNA fragment

Line up sticky ends

DNA ligase forms phosphodiester bonds between sugar-phosphate groups

71
Q

what are usually the vectors

A

Usually bacterial DNA / plasmids

72
Q

advantages of plasmids as vectors

A

Replicate independently

plasmid gets int host cell + combines with host DNA – form recombinant DNA

Have marker gene e.g antibiotic resistance – can tell if bacteria have taken up gene by growing in media containing antibiotic

73
Q

how can we tell the vector has taken up the plasmid

A

Plasmid – has second marker gene – show that plasmid contains recombinant DNA

Restriction enzyme inserts desired gene in this marker gene

If inserted right – marker gene wont function

74
Q

put the first 3 steps into a diagram

A
75
Q

what do you do after forming recombinant plasmid

A

transfer the vector

76
Q

what is transformation

A

Plasmid with recombinant DNA – inserted into host cell (bacteria)

77
Q

two methods to transfer the vector

A

calcium solution or electroporation

78
Q

transferring the vector - calcium

A

Culture bacterial cells + plasmids in calcium rich solution

Increase temp

Causes bacterial membrane to become permeable + plasmids enter

79
Q

transferring the vector - electroporation

A

Small electrical current applied to bacteria

Membranes – become very porous + plasmids move in

Also be used to get DNA fragments directly into eukaryotic cells

DNA – pass through cell + nuclear membrane

Control power or damage membrane

80
Q

how else can you make GM cells

A

electrofusion

81
Q

describe electrofusion

A

Tiny electric currents applied to membranes of 2 diff cells

Fuses cells + nuclear membranes

Forms hybrid / polyploid cell

Contains DNA from both

Produce GM plants

Animal cells – do not fuse easily – membrane have diff properties

Used in monoclonal antibodies

82
Q

how to form GM plants - M1

A

Agrobacterium tumefaciens – causes tumours in healthy plants

Desired gene placed in the Ti plasmid + marker gene (antibiotic resistance / fluorescence)

Carried into plant cell DNA

Transgenic plant forms callus

83
Q

what is a callus

A

mass of GM plant cells

84
Q

how do you test which bacteria have taken up the plasmid

A

expose host to an antibiotic that kills cells that lack the new genes

wait for surviving cells to form a callus

85
Q

how to form GM plants - M2

A

Electrofusion

Remove plant cell wall by cellulases

Electrofusion to form new polyploid cell

Plant hormones to stimulate growth of new cell wall

Callus formation

Transgenic plant formarion

86
Q

transgenic organism

A

organism contains nucleotide sequences from a different species

87
Q

why can we genetically modify

A

universal, meaning that almost every organism uses the same four nitrogenous bases

same codons code for the same amino acids in all living things (meaning that genetic information is transferable between species)

mechanisms of transcription and translation are also universal which means that the transferred DNA can be translated within cells of the genetically modified organism

88
Q

vectors list

A

Plasmids - transfer DNA into bacteria or yeast

Viruses - transfer DNA into human cells or bacteria

Liposomes - fuse with cell membranes to transfer DNA into cells

89
Q

what are restriction endonucleases

A

class of enzymes found in bacteria

used as a defence mechanism bagainst bacteriophages (viruses that infect bacteria)

e enzymes restrict a viral infection by cutting the viral genetic material into smaller pieces at specific nucleotide sequences within the molecule.

90
Q

summarise Genetic engineering into diagram

A
91
Q

why do we use the Ti-plasmid for plants

A

Soil bacterium infects plants by inserting the Ti-plasmid DNA into the plant genome

92
Q

Liposome

A

DNA is wrapped in a lipid molecule which can pass the lipid membrane by diffusion

93
Q

Getting the gene into the recipient cell

A

Microinjection - injecting the plasmid
Heat shock w/ calcium salts
Electroporation
Electrofusion

94
Q

describe heat shock w/ calcium salts

A

Reducing the temp to freezing and rapidly increasing to 40 degrees - increases permeability

Ca^2+ surrounds DNA (-ve), reduces repulsion, increases permeability

95
Q

Why do bacteria take up plasmds

A

Reproduce asexually - no genetic variation

Taking up plasmids from surroundings increases genetic variation, allows selection and evolution

96
Q

why do we like making recombinant proteins from eukaryotic cells rather than prokaryotic

A

these cells will carry out the post-translational modification - due to the presence of Golgi Apparatus

97
Q

advantages of GM to produce recombinant human proteins

A

More cost-effective to produce large volumes

Faster to produce

Reliable supply available

engineered to be identical to human proteins / have modifications that are beneficial

moral or ethical or religious concerns against using cow or pork produced proteins

Less allergic reactions

98
Q

Insulin

A

Bacteria plasmids modified to include human insulin gene

Inserted into E coli via transformation

Identified + isolated transgenic bacteria

Express human protein insulin

Extracted + purified

99
Q

advantages of GM plants / animals

A

Better than selective breeding

Organisms with the desired characteristics are produced more quickly

All organisms will contain the desired characteristic

The desired characteristic may come from a different species/kingdom

100
Q

uses of GM crops

A

Resistant to herbicides – increases productivity / yield

Resistant to pests – increases productivity / yield

Enriched in vitamins – increases the nutritional value

Golden rice

reduce the impact of farming on the environment due to there being less need to spray pesticides

101
Q

Insect resistance – soya

A

soya beans – susceptible to insect pests

genetically modified the already herbicide-resistant variety of soybean) by inserting a gene for the Bt toxin

gene is taken from the bacterium Bacillus thuringiensis

produce their own insecticide

insect ingests parts - alkaline conditions in their guts activate the toxin

the toxin is harmless to vertebrates as their stomach is highly acidic

killing the insect

BUT insect populations developed resistance

102
Q

uses of GM livestock

A

produce pharmaceutical drugs – pharming

biopharma sheep and goats have been genetically modified to produce a number of useful human proteins in their milk

the human blood protein known as AAT in sheep milk

the human protein antithrombin (stops blood clotting) in goat milk

103
Q

uses of GM pathogens

A

modified to shed light on their metabolism, drug resistance as well as how it causes damage to its host

aid research

act as vectors – infect cells – modified so can not replicate when inside host cell

104
Q

ethical issues of GM

A

Biotech companies charge farmers more money for GM seeds vs non-GM seeds to try and make back the money they have invested in their product

Seeds can not be kept from GM crops to regrow the crop the following year because GM crops do not “breed true”

Buying seeds year upon year can be a major struggle for farmers in developing countries – only buy from patent holder

lack of long-term research on the effects on human health

Organic farmers have complained that the pollen from GM crops may contaminate nearby non-GM crops that have been certified as organic

Environmentalists are concerned about the reduction in biodiversity for future generations

more vulnerable to extinction

Herbicide-resistance genes could transfer to weed plants resulting in “superweeds”

antibiotic-resistance genes that are commonly used as marker genes in genetic engineering could transfer to pathogenic organisms that would then be untreatable with antibiotics - “superbug”

Patenting – people in less developed countries prevented from using GM crops by patents // unable to afford

105
Q

pest resistance / disease resistance / herbicide resistance - perceived pros + cons

A
106
Q

Gene therapy

A

involves using various mechanisms to alter a person’s genetic material to treat, or cure, diseases

107
Q

somatic gene therapy

A
  • replacing mutant allele with healthy allele in affected somatic cell
108
Q

two types of somatic gene therapy

A

Ex vivo – the new gene is inserted via a virus vector into the cell outside the body. Blood or bone marrow cells are extracted and exposed to the virus which inserts the gene into these cells. These cells are then grown in the laboratory and returned to the person by an injection into a vein

In vivo – the new gene is inserted via a vector into cells inside the body

109
Q

germline gene therapy

A

insert healthy allele into germ cell – egg / embryo immediately after fertilisation

110
Q
A

Biology - Module 6 (6 decks)

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