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Module 5 > Manipulating genomes > Flashcards

Manipulating genomes Flashcards

1
Q

What is DNA sequencing

A

A technique that allows genes to be isolated and read

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2
Q

How is DNA cloned

A

1) Gene to be sequenced from a human is isolated using restriction enzymes from a bacterium
2) The DNA is then inserted into the bacterial plasmid (vector)
3) The plasmid is then inserted into the E-Coli host = when cultured it divides many times and the plasmid with the DNA insert will also be copied many times
4) Each new bacterium contains a copy of the gene
5) DNA is then isolated from the bacterium using restriction enzymes
6) The DNA is then made single stranded when the DNA is heated

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3
Q

Fred Sanger - DNA Sequencing is also called

A

End Termination

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4
Q

Fred Sanger - DNA Sequencing (experiment set up)

A

1) Used the single strand of DNA as a template for four experiments in separate dishes
2) Each dish contains a solution with the four bases + DNA polymerase

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5
Q

Fred Sanger - DNA Sequencing (experiment process)

A

3) To each dish a modified version of one of the DNA bases was added, which when incorporated into the synthesised complimentary strand of the DNA no more bases could be further added
- Each modified base was modified with a radioactive isotope
4) As the reaction progressed thousands of the DNA fragments of varying lengths were generated
5) DNA fragments were passed through a gel by electrophoresis
6) Smaller fragments travelled further so fragments could be sorted by length

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6
Q

Fred Sanger - DNA Sequencing

Because fragments were sorted into length order what did this convey

A

The nucleotide base at the end of each fragment can be read according to its radioactive label, which produces the complimentary base sequence to the DNA

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7
Q

The first sequencing machine

A

Was based on the Sanger method
-Used fluorescent dyes instead of radioactive isotopes
-Dyes glowed when scanned by a laser beam and the light signature was identified by the computer
-This method dispensed with the need for technicians to read autoradiograms

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8
Q

Autoradiogram

A

Photographs made when photographic films is exposed to molecules labelled with radioactivity

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9
Q

Primer

A

Used before DNA polymerase as DNA polymerase needs some DNA to attach too

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10
Q

Pyrosequencing - how is different from Sanger’s method

A

Uses sequencing by synthesis not by chain termination
= Synthesis of a single strand of DNA complimentary tot he strand being sequenced, whilst detecting by light emission, which base was added at each step

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11
Q

Pyrosequencing

(until primer is added)

A

1) A long length of DNA is cut into fragments of 300-800 base pairs using a nebuliser
2) These lengths are then degraded into single stranded DNA. These are the template DNAs and they are immobilised
3) Sequencing primer added

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12
Q

Pyrosequencing

(What is the DNA incubated with - step 4)

A

4) DNA incubated with the enzymes:
- DNA polymerase
-ATP sulfurylase
- Luciferase
-Apyrase

Substrates
-APS
-Luciferin
- One activated nucleotide ( ATP ; TTP ; CTP ; GTP)

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13
Q

What is an activated nucleotide

A

A nucleotide with two phosphate groups added to it

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14
Q

Pyrosequencing

(What happens after the DNA has been incubated)

A

5) One of the activated nucleotides (e.g. TTP) is incorporated into the complimentary strand of DNA using the strand to be sequenced as a template
6) The two phosphates are released as pyrophosphate
7) In the presence of APS, ATP sulfurylase converts the pyrophosphate to ATP
8) In the presence of ATP, luciferase converts luciferin to oxyluciferin
9) This conversion generates visible light, which is detected by a camera

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15
Q

Pyrosequencing

What does the amount of light generated indicate

A

The amount of light is proportional to the amount of ATP available and so indicated how many of the same type of the activated nucleotide was incorporated adjacently in the complimentary DNA strand
- More of the same base = more light

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16
Q

What happens to the unincorporated activated nucleotides

A

They are degraded by apyrase and the reaction starts again with another nucleotide

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17
Q

Electrophoresis

A

Process used to separate proteins/DNA fragments of different sizes
-Can separate fragments that differ from only one base pair

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18
Q

Outline electrophoresis set up

A

-Uses an agarose gel plate covered by a buffer solution
-Electrodes are placed in each of the tank so that when it is connected to the power supply an electric current can pass through
-DNA fragmented by restriction enzymes
-DNA has a negative charge due to many phosphate groups when placed in buffer solution at the cathode the fragments migrate towards the anode (positive electrode)
-Small fragments travel further (need to use a fixed time)

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19
Q

Dyes used

A

A loading dye to stain it - use a pipette to place in the buffer solution
-After buffer solution is poured away and a dye is added to the gel which adheres to the DNA and stains the fragments

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20
Q

Separating proteins

A

Similar to DNA but carried out in the presence of a charged detergent such as sodium dodecyl sulphate (SDS) which equalises the surface charge and allows the proteins to separate through the gel according to their molecular mass
- Proteins have a variety of R groups / bonds (all different charges)

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21
Q

What can separating proteins identify

A

-Sickle cell anaemia: haemoglobin S and not normal haemoglobin A
-Aplastic anaemia, thalassaemia, leukaemia higher then normal amount of fetal haemoglobin and lower amounts of haemoglobin A

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22
Q

What is the polymerase chain reaction (PCR)

A

A biomedical technology in molecular biology that can amplify a short length of DNA to thousands of millions of copies
-Used for forensic DNA analysis

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23
Q

What does PCR rely on

A

-DNA is made of two antiparallel backbone strands
-Each strand has a ‘5 end and a ‘3 end
-DNA only grows on the ‘3 end
-Base pairs pair up according to complementary base pairs A-T C-G

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24
Q

How does PCR differ from DNA replication

A

-Only short sequences of up to 10,000 base pairs of DNA can be replicated, not entire chromosomes
-Requires the addition of primer for the process to start
-A cycle of heating and cooling is needed to separate the DNA strands; bind primers to the strands and for the DNA to be replicated (cyclic reaction)

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25
Q

Why was initially the process of PCR quite time consuming

A

Cyclic reaction
-DNA heated to denature then cooled to 35 degrees to anneal the primers and allow the DNA polymerase to work
-Later DNA polymerase was obtained from the thermophilic bacterium - Taq polymerase which is stable at high temperatures (72 degrees is optimum)

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26
Q

Steps of PCR (till cooling)

A

1) Sample of DNA is mixed with DNA nucleotides; primers; magnesium ions (co-factors) and the enzyme taq polymerase
2) Mixture heated to 96 degrees to break the hydrogen bonds between complementary base pairs and denature the double stranded of DNA into two single strands
3) Mixture is cooled to 68 degrees so primers can anneal to one end of each single strand of DNA
=Small section of double stranded DNA at each end of single stranded DNA molecules

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27
Q

Steps of PCR

A

4) Taq DNA polymerase can bind to the end where there is double stranded DNA
5) Temperature raised to 72 degrees which keeps DNA single stranded
6) Taq polymerase catalyses the addition of DNA nucleotides starting at the end with the primer and proceeding in a 5’ 3’ direction
7) When Taw DNA polymerase reached the other end of the DNA molecules then a new double strand of DNA has been generated
8) Whole process is repeated

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28
Q

Steps of PCR- how does the DNA increase

A

Exponentially: 1- 2- 4 -8

After 5 repeats: 2(5)

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29
Q

Anneal

A

Bind by hydrogen bonds

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30
Q

Application of PCR

A

-Tissue typing
-Detection of oncogenes
-Detecting mutations
-Identifying viral infections
-Monitoring the spread of infectious disease
-Forensic science
-Research

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31
Q

Tissue typing (PCR)

A

Donor and recipient tissues can be typed prior to transplantation to reduce the risk of rejection of transplant

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32
Q

Detection of oncogenes (PCR)

A

If the type of mutation involved in a specific patients cancer is found then the medication may be tailored to that patient

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33
Q

Detecting mutations ( PCR)

A

A sample of DNA is analysed for the presence of a mutation that leads to a genetic disease
-Parents can be tested to see if they have a recessive allele for a particular gene ; fetal cells may be obtained for the mothers bloodstream for prenatal genetic screening; during IVF treatment one cell from an eight cell embryo can be used to analyse the fetal DNA before implantation

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34
Q

Identifying viral infections (PCR)

A

Sensitive PCR tests can detect small quantities of viral genome amongst the host cells DNA.
-This can be used to verify HIV

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35
Q

Monitoring the spread of infectious disease (PCR)

A

The spread of pathogens through a population of wild/ domestic animals / humans can be monitored and the emergence of new more virulent sub-types can be detected

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36
Q

Forensic science (PCR)

A

Small quantities of DNA can be amplified for DNA profiling to identify criminals or to ascertain parentage

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37
Q

The human genome project

A

A project in which scientists found that the human genome only contains about 24000 genes

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38
Q

Where are sequenced genomes stored

A

Gene banks

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39
Q

We share 99% of our genes with the chimpanzee what does this show

A

Genes that work well will be conserved by evolution

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40
Q

How was pig insulin used to treat patients with diabetes

A

It is similar to the human genes for insulin

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41
Q

What is FOX -P2

A

A change in a gene that allowed humans to speak

42
Q

How are organisms different

A

Not because their genes are completely different but because some of their shared genes have been altered and now in subtly different ways

-Some changes to the regulatory regions of DNA that do not code directly for proteins have also altered the expression of the genome - the regulatory and coding genes interact in such ways that without he number of genes being increased the number of proteins made may be increased

43
Q

How can the evolution of certain human pathogens be studied

A

Bacterial genomes can be extracted from ancient human bones

44
Q

How are humans different

A

Same genes but different alleles

45
Q

Substitution

A

A mutation which changes DNA sequences between humans

Place where substitutions occur are called SNP’s
-Some have no effect on the protein / alter the protein
-Some can alter the way RNA regulates the expression of another gene

46
Q

Methylation

A

Major role in gene regulation
-Can help researchers understand the development of certain human diseases i.e. why does cancer not develop in genetically similar individuals
- Study of this area is called epigenetics

47
Q

What does the researcher need to know to predict the amino acid sequence

A

-Have the organisms genome sequenced
-Know which genes code for a specific protein, by using knowledge of which base triplets code for which amino acid

=Determine the primary structure of an amino acid

Also needs to know which parts of the gene codes for introns/exons

48
Q

Synthetic biology

A

A science concerned with designing and building useful biological devices and systems
-Encompasses biotechnology, evolutionary biology, molecular biology, systems biology and biophysics
May be used to build engineered biological systems that store ad process information, provide food, maintain human health and enhance the environment

The sequencing of DNA found by analysing genomes provide potential building blocks for synthetic biologists to build devices

49
Q

Examples of synthetic biology applications

A

-Information storage
-Production of medicines
-Novel proteins
-Biosensors
-Nanotechnology

50
Q

Information storage - biotechnology application

A

Scientists can encode vast amounts of digital information onto a single strand of synthetic DNA

51
Q

Production of new medicines - biotechnology application

A

E-coli and yeast have both been genetically engineered to produce the precursor of a good antimalarial drug, artemisinin,
-Could only previously be extracted from plants at certain parts in their lifestyle

52
Q

Novel proteins - biotechnology application

A

Designed proteins have been produced e.g. one that is similar to haemoglobin and binds to 2 but not to carbon monoxide

53
Q

Biosensors -biotechnology application

A

Modified bioluminescent bacteria, placed on a coating of a microchip, glow if air is polluted with petroleum pollutants

54
Q

Nanotechnology - biotechnology application

A

Material can be produced for nanotechnology e.g. amyloid fibres for making biofilms - for functions such as adhesion

55
Q

Bioethics

A

Synthetic biology may raise issues with ethics and biosecurity
Due to using genetically modified organisms there are:
-Many advisory panels
-Scientific papers to manage risks

56
Q

What are short tandem repeats

A

Repetitive sequences of DNA that do not code for proteins
-May be 10/100 base pairs and all feature the same core sequence AACTG
-Occur at many places in the genome and may be repeated a random number of times
-Number of repeats = family resemblance

57
Q

DNA profiling procedure

A

1) DNA is obtained from the individual - either by a mouth swab, from saliva on a toothbrush, from blood or hair
2) DNA digested with restriction enzymes - cut the DNA into fragments, which will vary in size from individual to individual (these are the short tandem repeats and will be different dependant on the individual)
3) Fragments are separated by gel electrophoresis and stained - larger fragments travel the shortest distance in the gel
4) Banding pattern seen

58
Q

DNA profiling procedure (after banding pattern is seen)

A

5) DNA to which the individual is being compared to is treated with the same restriction enzymes and also subjected to electrophoresis
6) Banding patterns of DNA samples can then be compared

59
Q

Restriction enzymes

A

Cut the DNA at specific recognition sites

60
Q

Applications of DNA profiling

A

-Forensic science
-Maternity and paternity disputes
-Analysis of diseases

61
Q

Applications of DNA profiling - Forensic science

A

Been used too:
-Identify Nazi war criminals in South America
-Identify the remains of the Romanov family

62
Q

Applications of DNA profiling - maternity and paternity disputes

A

50% mother 50% father so STR 50/50
-Compare DNA profiles

63
Q

Applications of DNA profiling - analysis of disease

A

Protein electrophoresis can detect the type of haemoglobin and aid the diagnosis of sickle cell anaemia
-Huntington’s disease can also be detected

64
Q

Why does DNA profiling not look at genes that codes for proteins

A

Too similar

65
Q

What is a DNA probe

A

A short single-stranded length of DNA that is complimentary to the section of DNA being investigated

66
Q

What may the probe be labelled with-

A

1) A radioactive marker usually with the 32P on one of the phosphate groups on the probe strand. Once the probe has annealed (bound) by complimentary base pairing , to the piece of DNA, it can be revealed by exposure to photographic film by a fog mark

2) A fluorescent marker that emits colour on exposure to UV light.
-May also be used in automated DNA sequencing

67
Q

How are probes useful in locating specific DNA sequences

A

1) To locate a specific gene needed for use in genetic engineering
2) To identify the same gene in a variety of different genomes from different species when conducing gene comparison studies
3) To identify the presence/ absence of a specific allele for a particular genetic disease or gives susceptibility to a particular condition

68
Q

Microarrays

A

A number of different probes on a fixed surface
-Applying the DNA to the surface can reveal the presence of mutated alleles that match the fixed probes because the sample DNA will anneal to the complimentary fixed probes
-First split onto smaller fragments and amplified using PCR

69
Q

How can DNA mircroarray also be made

A

with fixed probes, specific for certain sequences in mutated alleles that cause genetic diseases, in the well

70
Q

Reference and DNA samples

A

Used as a control
-Labelled with fluorescent markers
-Reference and test subject both bind to a particular probe, the scan reveals fluorescence of both colours, which indicates the presence of a particular sequence in the test DNA

71
Q

Benefits of GM E-coli

A

GM micro-organisms can make human insulin to treat all diabetics, and human growth hormone to treat children with pituitary dwarfism

72
Q

Disadvantages of GM E-coli

A

Micro-organisms could escape into the wild and transfer marker genes for antibiotic resistance to other bacteria
However GM bacteria are also modified so they cannot synthesise an essential nutrient and therefore cannot live outside the lab

73
Q

Benefits of GM Plants (Bt Tobacco and Bt Maize)

A

Tobacco plants modified to produce a toxin normally produced by bacterium
-used as a pesticide as it is toxic to insects when inserted in plants reduces the need to spray it

74
Q

Disadvantages of GM Plants (Bt Tobacco and Bt Maize)

A

Bt toxic to monarch butterflies
-Do not take nectar from plants however as feed on milkweed so continues to thrive

75
Q

Benefits of GM soya beans

A

Resistant to herbicides so weeds that compete with the plant can be killed without killing the soya beans

76
Q

Disadvantages of GM soya beans

A

Risk of genes for herbicide resistance to pass into weeds
This produces ‘superweeds’
-Hasn’t happened yet

77
Q

Benefits of GM golden rice

A

Modified to contain beta carotene which helps children in India to not go blind

78
Q

Disadvantages of GM golden rice

A

Farmers would have to buy the seeds every year

79
Q

Benefits of GM plantains

A

Nutritionally enhanced to contain more zinc - helpful for people who are deficient as co-factor essential for regulation of insulin secretion

80
Q

Disadvantages of GM plantains

A

Foreign DNA may influence our own genes (all food we eat contain genes no different)

81
Q

Advantages of crop plants resistant to pests

A

Farmers don’t need to use pesticides
-Better for environment and health

82
Q

Disadvantages of crop plants resistant to pests

A

Farmers may not want GM seeds

83
Q

Advantages of GM pathogens

A

Viruses GM to have no virulence can be used to make vaccines as they still have the antigens on their surface
-Reduces the chance of the vaccine making the person ill
-Modified viruses can also be used as vectors in gene therapy

84
Q

Disadvantages of GM pathogens

A

In gene therapy allele may inserted into the genome in a way that increases the risk of cancer or interferes with gene regulation

85
Q

Advantages of GM mice

A

Useful for medical research and used to develop therapies for breast and prostate cancer
-Knock genes out so researchers can find the function of the genes

86
Q

Disadvantages of GM mice

A

Unethical - welfare of animals

87
Q

Advantages of GM pharmaceutical proteins

A

Can be inserted into goats/sheep and the human protein they express in their milk is harvest
-In a bacterial cell may be too large to synthesise

88
Q

disadvantages of GM pharmaceutical proteins

A

Welfare for GM sheep and goats

89
Q

Advantages of GM silk

A

Silk is a strong material but spiders silk is impossible to farm
-Genes for spider silk is inserted into goats, which they produce in their milk

90
Q

What is genetic engineering

A

Also known as recombinant DNA technology
-Involves combining the DNA of different organisms
-Genes are inserted into one organism, using suitable vectors

91
Q

Vector

A

Anything that can carry/insert DNA into a host organism

92
Q

Stages of genetic engineering

A

1) DNA is located using a DNA probe
2) Restriction enzymes cut out the desired gene
3) Enzymes insert gene into DNA vector, which is taken from the bacterium (recombinant DNA) (DNA ligase - sticks it in)
4) Bacteria reproduce - creating a large number of bacteria with the new characteristic

93
Q

Different ways of obtaining the required gene

From mRNA

A

1) mRNA can be obtained from the cells when the gene is being expressed. An enzyme reverse transcriptase can catalyse the formation of a single strand of DNA using mRNA as a template
- Addition of primers and DNA polymerase can make cDNA into double stranded DNA whose bases code for the original protein

94
Q

Different ways of obtaining the required gene

A

2) If scientists know the nucleotide sequence of the gene, then the gene can be synthesised using an automated polynucleotide synthesiser
3) If they know the sequence of the gene they can use PCR to amplify the gene from the genomic DNA
4) A DNA probe can be used to locate a gene within the genome and the gene can then be cut out using restriction enzymes

95
Q

Placing a gene in a vector

A

1) Plasmids can be obtained from organisms such as bacteria mixed with restriction enzymes that will cut the plasmid at specific recognition sites
2)The plasmid has exposed unpaired nucleotide bases called sticky ends because of how its been cut
3) If the free nucleotide bases, comp to the sticky ends of the plasmid, are added to the ends of the gene to be inserted, then the gene and cut plasmid should anneal (bind)
4) DNA ligase enzymes catalyse this annealing

96
Q

Another way of placing a gene in a vector

A

A gene may be sealed into a weakened virus that could carry it into a host cell

97
Q

Getting the vector into the recipient cell

Heat shock treatment

A

DNA does not easily cross the recipients plasma membrane

1) Heat shock treatment: If bacteria are subjected to alternating periods of cold (0 degrees) and heat (42 degrees) in the Prescence of calcium chloride, their walls will become more porous and allow in the recombinant vector

=Positive calcium ions surround the negatively charged parts of both the DNA molecules and phospholipids in the cell membrane, thus reducing repulsion between the foreign DNA and the host cell membrane

98
Q

Getting the vector into the recipient cell

A

2) Electroporation - a high voltage pulse is applied to the cell to disrupt the membrane

3) Electrofusion - electrical fields help to introduce DNA into cells

4) Transfection - DNA can be packaged into a bacteriophage (viruses that infect and replicate in bacterial cells) which can then transfect the host cell

99
Q

Getting the vector into the recipient cell

A. Tumefacien

A

T1 (recombinant) plasmid A. Tumefacien, which infects some plants and naturally its genome into the host cells genome

100
Q

Getting the vector into the recipient cell

What happens if plant are not susceptible to A. Tumefacien

A

Direct methods can be used
Small pieces of gold/tungsten are coated with DNA and shot into the plant cells
-This is called a ‘gene gun’

101
Q

Reverse transcriptase

A

Retroviruses, such as HIV, which contain RNA that they inject into the host genome, have reverse transcriptase enzymes that catalyse the production of cDNA (comp DNA) using RNA as a template
-Reverse of transcription

102
Q

Restriction enzymes

A

Bacteria have to protect from attack by phage viruses
-Cut up foreign viral DNA, which prevents the virus from making copies of itself
-Prokaryotic DNA is protected from the action of these endonucleases by being methylated at the recognition sites

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