Manipulating Genomes Flashcards

1
Q

What is the PCR?

A

method for amplifying DNA fragments rapidly and effectively

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2
Q

What are primers?

A

Short sequences of nucleotides that attach to the start and end of a DNA fragment to be copied

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3
Q

what components are required in PCR?

A

DNA fragment

Primers

Taq polymerase

Free nucleotides

Thermocycler

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4
Q

What is Taq polymerase?

A

A type of DNA polymerase from thermophilic bacteria

can withstand high temperatures without denaturing

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5
Q

What is stage 1 of PCR?

A

Separation

Temp: 95 degrees celsius

DNA is heated to separate the 2 strands by breaking the hydrogen bonds

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6
Q

What is stage 2 of PCR?

A

Annealing (adding primers)

Temp: 55 degrees Celsius

Primers attach to the specific start and end points of the separated DNA strands

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7
Q

What is stage 3 of PCR?

A

DNA synthesis (extension)

Temp: 72 degrees Celsius

Taq polymerase adds free nucleotides to the target strands to form a complete copy of the strand

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8
Q

Advantages of the PCR

A

Rapid speed

Precision

Only small samples of DNA needed

No cells needed

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9
Q

What is gel electrophoresis?

A

technique used to separate DNA based on size using an electric current applied to an agarose gel matrix

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10
Q

How is gel electrophoresis set up?

A

Insert a gel tray with solid agarose gel into a gel tank

Ensure walls are close to negative electrode to position gel correctly

Pour buffer solution over gel until submerged

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11
Q

How are samples loaded in electrophoresis

A

Mix DNA samples with a dye to make them visible

Micropipette equal volumes into the wells

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12
Q

What happens during electrophoresis

A

A voltage is applied across the Gel

Fragments of DNA move toward the positive electrode

Smaller fragments travel faster

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13
Q

What are the key stages of gene transfer?

A

Desired gene is identified and isolated

Multiple copies made using PCR

The genes is inserted into vector

Vector delivers gene into cells in different organism

Cells with new gene are identified using marker genes

Cells with new genes are cloned

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14
Q

How are DNA fragments produced

A

mRNA is extracted from cells

mRNA is reverse transcribed by reverse transcriptase and free nucleotides to form cDNA

Free floating nucleotides and DNA polymerase are used to form the other strand of cDNA

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15
Q

What are restriction enzymes

A

recognize and cut DNA at specific palindromic nucleotide sequences to isolate gene fragments

palindromic nucleotides read the same forward and backward

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16
Q

How do restriction enzymes work?

A

DNA incubated with chosen restriction enzyme

Restriction enzymes identify palindromic sequences and cut DNA if their recognition sequence is present

Recognition sequences are at the beginning and end of desired DNA fragment

Enzymes cut target gene fragment out by hydrolysis

17
Q

What are sticky ends?

A

Short overhanging sequences of unpaired bases that can bind to other DNA fragments

18
Q

How are DNA fragments inserted into vectors

A

Vector is cut open at specific site using restriction enzyme - creates sticky ends

Same restriction enzyme is used to cut target DNA fragment to make complementary sticky ends

DNA ligase forms phosphodiester bonds, joining the sticky ends of the vector and DNA fragment

Recombinant DNA formed

19
Q

How is recombinant DNA transferred into host cell using plasma vectors?

A

Host cells are treated to enhance uptake of plasmids that have recombinant DNA

Adding calcium ions and temperature shifts increases membrane permeability to plasmids

Electroporation uses an electrical current to make bacterial membranes more porous

helps plasmids enter

20
Q

How is recombinant DNA transferred into host cell using bacteriophage vectors?

A

Bacteriophages inject their DNA into host bacterial cells during infection

Recombinant DNA is injected into host cell

21
Q

How can transformed host cells be identified?

A

Marker genes indicate which host cells have recombinant DNA

Inserted into vectors alongside target genes

transformed cells are cultivated on selective agar plates

Only transformed cells are marked

Transformed cells are cultured to be mass produced

22
Q

What are the different types of marker genes?

A

Marker gene for a specific trait

Marker gene that is visible under UV light

Marker gene coding for a specific enzyme that changes the colour of a specific substrate

23
Q

How can gene therapy be used to treat genetic disorders?

A

Identify abnormal gene creating the disorder

Engineer a functional version of this gene

Deliver normal allele to nucleus of target cell using vector

Ensure gene has correctly integrated into cells DNA

24
Q

Explain counteracting recessive disorders

A

Add functional dominant alleles

This silences non-functional recessive alleles

25
Explain silencing faulty dominant alleles
Insert DNA sequences that inactivate harmful dominant alleles This prevents dominant allele from functioning properly
26
Somatic vs germline gene therapy: Definition
Somatic: Replacing mutant alleles with healthy alleles in affected somatic cells Germline: Inserting healthy allele into germ cells or embryos to prevent genetic disease from birth
27
Somatic vs germline gene therapy: Target cells
Somatic: Alters somatic cells affected by disease Germline: Alters germ cells or embryos
28
Somatic vs germline gene therapy: Tissues or organs affected
Somatic: Impacts specific tissues and organs only Germline: Influences all cells within the body
29
Somatic vs germline gene therapy: Inheritance
Somatic: Genetic modifications are not inherited by offspring Germline: Modifications can be passed down to future generations
30
Ethical benefits of gene therapy
Extends life by treating disease Enhances quality of life Reduces overall disease burden in population
31
Ethical concerns of gene therapy
Risk of causing unintended harm High cost which restricts access Potential misuse for aesthetics
32
Issues with somatic gene therapy
Delivering health alleles into the nucleus of a cell is challenging Maintaining the expression of healthy alleles is challenging Effects are short term as somatic cells have a limited lifespan
33
Issues with germline gene therapy
Unborn child cant provide consent Causes irreversible changes Could be used for aesthetic and not medical reasons
34
Outline the process of DNA sequencing
DNA mixed with primers, DNA polymerase, nucleotides and terminator bases DNA split and copied multiple times DNA polymerase adds nucleotides to rebuild new DNA strands Terminator added to stop DNA synthesis and is tagged by fluorescent color This produces DNA fragments of all possible lengths DNA fragments are separated by length Laser detects fluorescent colors of terminator bases in each fragment to determine sequence order