Biology > Manipulating Genomes > Flashcards

Manipulating Genomes Flashcards

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1
Q

What is the sanger method?

A

-Original method of DNA sequencing
-Involves using marked terminator bases, PCR and electrophoresis

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2
Q

Define human genome.

A

All of the genetic material it contains

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3
Q

Define Exons.

A

A section of DNA that codes for amino acids and can be used to form the mature mRNA.

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4
Q

Define Introns.

A

Non coding segments of DNA which gets spliced out to form a mRNA to code for a protein,

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5
Q

What are VNTRS?

A

-Variable number tandem repeats sequences of bases in introns
-Unique to each person
-Also called STRs (short tandem
repeats

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6
Q

What is a mini satellite/VNTRS?

A

Short DNA sequences that vary in number between individuals- 20-50 base pairs.

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7
Q

What are micro satellite/STRS?

A

Short, repeated DNA sequences scattered through out the genome.

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8
Q

Define DNA profiling.

A

Producing an image of the patterns in the DNA of an individual.

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9
Q

What are the 5 steps to producing a DNA profile?

A
  1. Extracting a DNA profile
    2.Digesting the sample
    3.Separating the DNA fragments
    4.Hybridisation
    5.Seeing the evidence
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10
Q

Where do mini satellite appear on the chromosome?

A

The satellites always appear at the same positions on the chromosomes but the number of repeats varies with individuals

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11
Q

Where does the mini satellite appear on the chromosome?

A

Because they occur at more than 1000 location

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12
Q

How does extracting the DNA profile occur in the first stage of producing a DNA profile?

A

DNA must be extracted from a tissue sample.
PCR can be used to develop enough DNA for a profile.

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13
Q

What happens during Digesting the sample in the second stage of producing a DNA sample?

A

Using RESTRICTION ENDONUCLEASES the DNA strands are cut into small fragments.
The enzyme makes 2 cuts- 1 through teach strand of the double helix
Leaves satellites intact.

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14
Q

Define restriction site?

A

Specific cut at a DNA nucleotide sequence.

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15
Q

What happens during separating the DNA fragments during the third stage of producing a DNA profile?

A

Thee cut fragments of DNA needs to be separated to form a clear and recognisable pattern.

Gel electrophoresis occurs
-The gel is soaked in alkali to separate the DNA strands into single strands.

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16
Q

Define gel electrophoresis.

A

A technique which uses an electric current to move DNA fragments through a gel medium for charged particles to move.

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17
Q

What happens during hybridisation at the 4th stage of producing a DNA profile?

A

Florescent DNA probes are added to the DNA fragments these Florescent DNA probes are short and complementary strands of DNA and bind to the sequence under optimum conditions.
-The excess probes are washed off.

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18
Q

What happens during the last stage of DNA profiling- seeing the evidence?

A

Florescent DNA is placed under a UV light so tags glow and fragments are given bars of pattern this is the DNA profile.

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19
Q

Define high put sequencing.

A

-Modern methods of DNA sequencing that are faster than the Sanger method
-More automated than the Sanger
method

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20
Q

Define reverse transcriptase.

A

An enzyme that makes cDNA
(single-stranded copies of
DNA)from mRNA

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21
Q

Define Restriction endonuclease.

A

-Enzymes that cut up DNA to create fragments
-Cut at specific recognition/restriction sequences
-Results in sticky ends

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22
Q

What is gel electrophoresis?

A

-Separation of DNA samples using
an electrical voltage

-Different lengths of DNA are
separated

-The smallest/shortest/lightest
pieces of DNA move the furthest

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23
Q

Why does the DNA fragments move during gel electrophresis?

A

-DNA is negatively charged and
moves towards the positive end of the gel

-The shorter the piece of DNA, the
faster and further it moves

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24
Q

What are the steps of Gel electrophoresis?

A

1.DNA loading dye is added to PCR tubes( contains differnt samples) then pipetted into wells of the agarose gel plate

  1. Electric current is applied across the gel plate.
    DNA moves to positive anode

3.Gel is left running for many hours but can be sped up by a increasing the voltage.

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25
What is PCR?
-Fragments of DNA can be amplified (copied) via the polymerase chain reaction (PCR). -This is done by an automated machine called a thermocycler.
26
What are the uses of PCR?
-Used widely in gene technology to make large numbers of copies of DNA fragments e.g. forensics, genotyping, cloning, paternity tests
27
What is the PCR process?
1. Denaturation-Increase temperature to 95 C to break hydrogen bonds & split DNA .into single strands 2. Annealing- Temperature decreased to 55 C so primers can attach DNA polymerase joins complementary nucleotides & makes a new strand 3. Temperature increased to 72 C (optimum for Taq DNA polymerase)
28
How to view electrophoresis?
The agar gel will shrink and crack as it dries, and therefore it can be transferred to a nylon sheet. The nylon sheet can then be exposed to x- rays (if the DNA was marked with a radioactive tag), or to UV light (if the DNA was marked with a fluorescent tag).
29
What are the differences between DNA electrophoresis and Protein Electrophoresis?
The method is very similar to that for DNA with two differences: As proteins have a complex 3D shape, they need to be denatured first, this is done by heating the proteins. All proteins need to be made to have a negative charge so that they move through the gel, this is done by using a chemical called sodium dodecyl sulphate (SDS).
30
What is need for PCR?
Thermocycler DNA fragment to be amplified DNA polymerase – Taq polymerase Primers DNA nucleotides
31
Define DNA sequencing?
The process used to determine the precise sequencing of nucleotides in a length of DNA
32
What are chain terminating nucleotides used for?
They are used to interrupt PCR and terminate DNA synthesis.
33
What is the process of the sanger method?
1. Single stranded DNA is used to template in 4 tubes which contain **primers, DNA polymerase, free nucleotides**. The complementary strand is being created. 2. In tubes one CT nucleotide are added. All tubes added to thermocycler. 3. PCR 4.DNA polymerase binds to template DNA and synthesises a complimentary strand. 5.PCR cycle is repeated until another CT is added 6. Gel electrophoresis is used to separate the fragments.
34
What is the sanger method used for?
It is used for validating gene mutations, sequencing of small organism etc .
35
Define pyrosequencing
A method of DNA sequencing detecting light emitted during enzymatic incorporation of nucleotides. 'sequencing by synthesis'- next generation sequencing.
36
What are the steps to pyrosequencing?
1. DNA molecules is broken up into fragments 2. DNA fragments are separated into strands and act as the template- fixed to a flow cell 3.Flow cell has millions of DNA strand fixed to their own well 4.ssDN is incubated with sequencing primers, enzymes and nucleotides. 5.One of the activated nucleotides are added at a time and light generated detected- 2 phosphoyl group is released as pyrophosphate.
37
What happens when the enzymes of Pyrosequencing are used?
6. Pyrophosphate+ APS = ATP catalysed by ATP sulfurylase 7.Luciferase uses ATP to convert Luciferin-> oxyluciferin to generate visible light which is detected by a camera. 8.Amount of light generated is proportion to amount of ATP ( non incorporated nucleotides are degraded by apyrase) 9. AA differnt activated nucleotide is washed across the flow cell resulting in another flash Cycle repeated till all DNA is synthesised.
38
What Enzymes are used in Pyrosequencing?
Luciferase ATP sulfurylase Apyrase
39
Define synthetic biology.
-The creation of artificial pathways, organisms, devices or the redesign of natural systems
40
What has sequencing of genomes allowed us to do?
Sequencing the genomes has enabled the development of synthetic biology
41
What has developments in synthetic biology allowed us to do?
-Genetic engineering: Insulin producing bacteria -The use of biological systems in industry: immobilised enzymes - The synthesis of new genes to replace faulty versions of genes: replacing the faulty gene that causes cystic fibrosis -The synthesis of new organisms: new bacterial genomes have been created by scientists)
42
What is bioinformatics?
The use of software to analyse, organise and store biological data. -This includes databases storing all known alleles, amino acid sequences of proteins and structures of proteins.
43
What is computational biology?
The use of computers to study biology e.g. simulations, models and algorithms
44
What is DNA barcoding?
A method of identifying species using a short standardised DNA sequence from a specific gene so comparison can be made between species.
45
Define genome analysis.
Process of sequencing, mapping and annotating genomes to understand its functional elements.
46
What is proteomics?
The study of entire amino acid sequencing of an organism.
47
What is genomics?
The study of an organisms genome and genes, function interactions and structure.
48
What is a proteome?
A collection of all the proteins produced by the genome in a cell/organism which is determined by working out the base sequence of every gene.
49
What is a splicesomes?
A RNA machine in eukaryotic cells that edits mRNA by removing noncoding introns and joins coding exons creating a mature mRNA ready for proteins synthesis.
50
Define plasmid.
-A small loop of bacterial DNA -Contains only a few genes -Contains the genes for antibiotic resistance
51
Define recombinant plasmid.
A small loop of bacterial DNA with the DNA from another organism inserted into it
52
Define transgenic organism.
A living organisms whose DNA has been altered by inserting genes from a different species.
53
How can a transgenic organism express this gene?
As the genetic code and mechanism is universal.
54
Define genetic engineering.
Manipulating an organisms genome to achieve a desired outcome.
55
What are the three ways of isolating a desired gene?
-Converting RNA to DNA using reverse transcriptase -Using Restriction endonucleases -Using a polynucleotide synthesiser
56
How do you isolate a desired gene by converting RNA to DNA by reverse transcriptase?
1.Cells that express the desired genes produce mRNA 2.mRNA is used as a template for synthesis of a cDNA 3.T get rid of mRNA the mRNA is hydrolysed with an enzyme to isolate cDNA 4.Double sDNA is synthesised by DNA polymerase- cDNA is the template. 5.Double stranded DNA contains code for desired protein.
57
What are the advantages of converting RNA to DNA using reverse transcriptase as a method?
-Introns has been spliced out of the gene -Allows for gene to correctly transcribed and translated by prokaryotic cells.
58
How do you isolate a desired gene using restriction endonuclease?
Restriction endo nuclease cutes up DNA at a specific sequence at recognition sites. Each enzyme cutes at differnt sites some enzymes create clean cuts some create sticky ends.
59
What are sticky ends?
-Exposed staggered ends of bases Palindromic base sequences -Created by restriction endonuclease enzymes.
60
What is palindromic sequence?
Sequences of bases that read the same forwards as they do backwards
61
What is the blunt end?
-When a restriction endonuclease cuts the DNA double-strand in the same position -There is no overhang of bases
62
How is the gene isolated using a polynucleotide synthesiser?
1.Desires sequence can be determined by the primary structure of the protein chosen 2.Determined polynucleotide sequence is fed into a computer which helps deign short sequences of nucleotides which overlap- oligonucleotides. 3. Short sequences are synthesised by machine in an automated process. 4.Gene assembled by joining oligonucleotides then amplified using PCR 5.Poly nucleotide is checked for errors.
63
What are the advantages of using a polynucleotide synthesiser for isolating the gene
-Gene not containing introns or other non coding DNA -Allows for it to be correctly transcribed/translated.
64
How are is the desired gene inserted into the vector?
1.Restriction endonuclease cut the plasmid at a specific recognition site. 2.DNA from gene of the desired gene anneals the plasmid 3.Gene is permanently incorporated into the plasmid by the action of DNA ligase 4.Desired gene is inserted into plasmid which creates a recombinant plasmid.
65
Why must the same enzyme cutting the desired gene out of the DNA strand the same one cutting the vector?
As they need to cute the same complementary restriction site.
66
Ways to transfer the recombinant plasmid into the host cell?
-using heat shock treatment -transformation by electroporation -electrofusion -transfection
67
How does heat shock treatment allow the plasmid to pass through the cell membrane of a bacteria cell?
1) Mix bacteria and plasmid in a solution which contains CaCl2 in a test tube. 2.Test tube is placed in 0 degrees ice for a certain amount of time->42 degrees ->0 degrees 3. Increase of temperature creates pores in membrane.
68
Why is CaCl2 added to the heat shock treatement?
Plasmids need Ca+ ions for plamids to pass through the membrane. Ca+ reduces the repulsion of the DNA and the membrane.
69
What is electroporation?
When an electrical current is applied to the membrane to make it more porous
70
What is electrofusion?
Electrical fields are used to introduce DNA into the cells
71
What is transfection?
DNA is packaged into bacteriophages which can enter host cells and release DNA.
72
What is the history of ethical concerns of gene technology?
Initial concerns of human genes into microorganisms but it has seen has beneficial by most people as it has been used for years. -still concerns for biological warfare.
73
What are the uses for gene therapy micro organisms?
Vaccines Insulin DNA libraries Antibiotics Enzymes
74
What are ethical concerns for GM plants?
-Non pest insects can be damaged -Antibiotic genes can spread to wild populations and spread antibiotic resistance -transferred genes can mutate -reduction of biodiversity -allergies to proteins in GM crops.
75
What are some genetically engineered characteristics into plants?
Pest resistance disease resistant Herbicide resistance Extended shelf life Growing conditions Nutritional value Medical Uses
76
What are the uses of GM animals?
-Swine fever- resistance pigs -Faster growing salmon
77
What are some ethical concerns of GM animals?
-creating animal models which create cancer in mice from deleted genes -Side effects -Ethical issues- no consent -might die during development.
78
What are some uses of GM in humans?
Gene therapy -Some disease are a result of mutant genes -Reduces the need for surgery/meds
79
What is a transformation?
-The process of getting a plasmid to re-enter a bacterium -Involves calcium ions and temperature shocking
80
How can transformed cell be identified?
Using marker genes Antibiotic resistance genes Genes coding for fluorescent proteins Genes coding for enzymes
81
What are marker genes?
Genes on the plasmid used to identify which bacteria successfully took up the recombinant plasmid
82
What is pharming?
Genetic engineering to produce medicinal products.
83
How is pharming carried out?
This is when a gene is inserted into another organism so that they produce a human protein. e.g. insulin-producing bacteria e.g. goats producing human alpha antitrypsin for emphysema treatment
84
What is gene therapy?
Gene therapy is when human DNA is altered to treat disorders Two types: somatic cell gene therapy germline gene therapy
85
What is somatic cell gene therapy?
Altering the alleles in body (somatic) cells It is also not permanent as when these cells are destroyed, the inserted alleles are lost
86
Why is somatic cell gene therapy a treatment not a cure?
This is a treatment not a cure for the disease as it only changes the DNA in a small number of cells
87
Why is somatic cell gene therapy not inherited?
It is also not permanent as when these cells are destroyed, the inserted alleles are lost
88
What is germ line therapy?
The alteration of the DNA in the cells giving rise to the gametes or in the zygote so that the offspring would not inherit the faulty allele -All cells in the developing organism are genetically altered
89
What are the ethical concerns of germ line therapy?
-Designer babies -human rights of consent -Unknown impact on individual
90
What are the ethical concerns of somatic cell therapy?
-Unknown long term impact -In equality of access- expensive.
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