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Manipulating genomes Flashcards

1
Q

What is a DNA profile and how are they constructed?

A

A DNA profile is a genetic fingerprint that is unique to each person (except identical twins) and they are constructed using techniques such as the polymerase chain reaction (PCR) and electrophoresis

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2
Q

How is a DNA profile produced?

A

DNA extracted (and many copies made using PCR)
Digested (broken into fragments) using restriction endonucleases
Separated using electrophoresis
Hybridised with probes (bind to fragments and enable them to be visualised
Visualised in banding patterns (bars)

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3
Q

What is PCR and how is it used in DNA profiling?

A

Polymerase chain reaction - DNA amplification (copying).
DNA sample is placed in a thermocycler, which cycles through 3 temperatures:
95°C - breaks hydrogen bonds in the DNA, splitting it into two strands
55°C - primers bond to the end of each DNA strand
72°C - taq DNA polymerase joins free nucleotides to each strand

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4
Q

What is electrophoresis and how is it used in DNA profiling?

A

Separation of DNA fragments - DNA fragments are placed at the end of a gel plate. A positive electrode is at the opposite end of the plate
DNA moves towards the positive electrode when a current is applied (because all DNA fragments have phosphate groups with negative charges)
Longer fragments move slower, shorter fragments move faster
DNA fragments therefore separated into bands based on size

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5
Q

What are restriction endonucleases?

A

Enzymes found in bacteria - more than 50 are known, and each one cuts DNA at specific base sequences (recognition sites). As well as digesting DNA prior to electrophoresis, they are used for genetic engineering

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6
Q

Why is taq DNA polymerase used, rather than human DNA polymerase?

A

It is obtained from a thermophilic bacterium (Taq) so this form of polymerase is tolerant to heat so does not denature during temperature cycling. Also easier to obtain and potentially more ethical than using human DNA polymerase.

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7
Q

What are VNTRs?

A

Variable number tandem repeats. DNA profiles are often formed from these sections of DNA as the patterns of VNTRs differ between people - there is a very low probability of two unrelated individuals having the same VNTR profile.

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8
Q

What are 3 uses of DNA profiling?

A

In forensics (legal applications) - such as criminal investigations and paternity testing
Disease risk analysis
Classification

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9
Q

How is DNA sequenced?

A

PCR is conducted
However, some of the free nucleotides in PCR have been modified in two ways:
- when they bond to a DNA strand they terminate polymerisation
- They are fluorescently coloured - A, T, C and G have different colours
New DNA strands stop growing whenever a terminator base is added - PCR is interrupted
This results in every possible chain length being produced
Lasers detect the final base on each chain
The sequence of DNA bases can therefore be worked out

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10
Q

How are DNA sequences used?

A

Disease analysis - particular gene variants can be sequenced and linked to the risk of inheriting certain diseases. Sequencing pathogen genomes enables identification of antibiotic-resistant bacteria, pinpointing genetic markers for vaccines, and identification of targets for drugs
Classification - identifying species by using DNA barcodes. Studying evolutionary relationships by comparing similarities and differences between species’ base sequences
Genotype-phenotype relationships - amino acid sequences do not always match those predicted from base sequences - several phenotypes are possible from the same genotype - knowledge of both amino acid and base sequences enables comparisons to be made
Synthetic biology - genetic engineering requires knowledge of base sequences

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11
Q

What is the difference between bioinformatics and computational biology?

A

Bioinformatics is a toolkit - creation of databases and computer software that can be used to solve biological questions. Computational biology is the application of bioinformatics - e.g. sequencing of genomes relies on bioinformatics and is therefore an example of computational biology.

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12
Q

What is the difference between DNA profiling and DNA sequencing?

A

DNA profiling produces a genetic fingerprint, unique to an individual, which is based on particular sections of DNA.
DNA sequencing determines the precise base sequences in DNA

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13
Q

What is genetic engineering?

A

Altering genes and transferring them between species

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14
Q

What are the 2 ways a desired gene can be extracted?

A
  • Producing the gene from an mRNA template

- Cutting the gene out using restriction endonucleases

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15
Q

Describe the use of reverse transcriptase in genetic engineering

A

mRNA (transcribed from desired gene) extracted from cells. Reverse transcriptase is used to convert mRNA to cDNA (a single strand of complementary DNA)

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16
Q

Describe the use of restriction endonucleases in genetic engineering

A

Gene is cut from its source DNA - cutting the gene and plasmid with the same enzyme produces two sets of sticky ends with complementary base pairings, enabling the gene to be inserted into the plasmid

17
Q

What is recombinant DNA?

A

DNA combined from two different sources

18
Q

What are plasmids?

A

Small circular pieces of DNA found in prokaryotic cells

19
Q

What are sticky ends?

A

two short sequences of exposed, unpaired bases at ends of DNA that has been cut

20
Q

What are marker genes?

A

Indicate whether or not a gene (and therefore a plasmid) has been successfully taken up by bacterial cells. Marker genes are usually for antibiotic resistance or fluorescence

21
Q

What is used to transfer the gene into the organism that is being modified?

A

A vector

22
Q

Give two examples of vectors:

A

Plasmids - circular bacterial DNA; by far the most common vector. Bacterial artificial chromosomes (BACs) are synthetic structures based on plasmids. DNA ligase is used to seal the gene into the plasmid
Viruses (e.g. bacteriophages, which naturally infect bacterial cells)

23
Q

What is the process of transferring the donated gene into the recipient’s cells called?

A

Transformation

24
Q

Describe the culture heating method of transformation

A

Bacterial cell membranes become more permeable when heated in a calcium-rich solution. Plasmids are then able to diffuse into the cells

25
Q

Describe the electroporation method of transformation

A

Electric current disrupts the cell membrane, enabling plasmids to enter

26
Q

Describe the electrofusion method of transformation

A

Electric currents enable the cell and nuclear membranes of two different cells to fuse - used in plants

27
Q

Describe the viral transfer method of transformation

A

Viruses naturally infect cells, and this mechanism can be exploited to insert DNA directly into target cells

28
Q

Describe the Agrobacterium tumefaciens infection method of transformation

A

This bacterium naturally infects plant cells and can be used to introduce recombinant plasmids

29
Q

What are the potential ethical issues with genetically modified microorganisms?

A

The use of the technology for biological warfare

30
Q

What are the potential ethical issues with pest resistance in plants?

A

GM plants could produce toxins that might harm insects other than targeted pests, and also the pests may require the plants to survive

31
Q

What are the potential ethical issues of pharming (producing human medicines from GM animals)?

A

Is animal welfare compromised? Will genetic engineering damage the health and livilhoods of the animals?

32
Q

What are the potential ethical issues of patenting (i.e. legal ownership of GM technology)?

A

How available will technology be for those it might benefit? Companies are able to patent techniques and GM organisms, and farmers in poor countries are forced to pay for their use.
Making money put above welfare of people, e.g. with diseases or conditions that could be treated

33
Q

What is gene therapy?

A

Addition of beneficial alleles to the cells of people with disease-causing alleles

34
Q

Describe somatic cell therapy

A

Human body cells targeted - haemophilia, cystic fibrosis, immune diseases treated with this type of gene therapy.
Limitations:
- There are possible risks of additional health problems
- Introduced genes are non-inheritable
- requires repeat treatments

35
Q

Describe germline therapy

A

Gametes or early embryonic cells are targeted. No current examples - illegal in humans - ethical issues - changes are permanent, and who decides which genes are targeted?

36
Q

How can genes be transferred into target cells?

A

Using either harmless viruses or liposomes (hollow spheres of lipid molecules)

37
Q

What enzyme is used to seal the gene into the plasmid in genetic engineering (producing recombinant DNA)?

A

DNA ligase

Biology (11 decks)

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