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Bio Yr12 > 11. DNA manipulation > Flashcards

11. DNA manipulation Flashcards

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Biology 101 flashcards
1
Q

What is biotechnology and what is it important for?

A

Using living things to make new products or systems.

-Important for biotechnology, agriculture, the environment, and medicine

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2
Q

What is genetic engineering and what are GMOs?

A

Manipulation of the normal function of genes to achieve the desired outcome.

  • Modern biotechnology often falls under the definition of genetic engineering
  • Organisms that have had their genes altered using these methods are genetically modified organisms (GMO)
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3
Q

List the 3 types of GMOs:

A
  • Knock-out organisms
  • Knock-in organisms
  • Transgenic organisms
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4
Q

What do knock-out organisms involve?

A

Cutting out a gene or part of a gene to prevent expression or normal functioning of a gene product.

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5
Q

What do knock-in organisms involve?

A

Adding a reporter gene in front of a promoter of an unknown gene to learn where the gene is expressed.

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6
Q

What do transgenic organisms involve?

A

Genes added from other organisms to change the organism’s characteristics.

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7
Q

What is PCR (polymerase chain reaction)?

A

An in vitro (in glass) method of making many identical pieces of a section of DNA, where each cycle doubles the number of copies.

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8
Q

List the components required for PCR:

A
  • DNA/Taq polymerase (from a hot spring bacteria Thermus aquaticus)
  • DNA template
  • Primers (short sequence of DNA complementary to a section of the template DNA)
  • Free nucleotides
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9
Q

Describe the 4 stages of PCR:

A
  1. Denaturing: DNA is heated to above 93C to separate the two DNA strands.
  2. Annealing: The temperature is cooled to allow primers to anneal (bind) to their complementary sequences on each of the two strands of DNA (at the 3’ end of the DNA template strand).
  3. Extension: Temperature increase to 70C as the optimum temperature of the enzyme, Taq DNA Polymerase. This enzyme binds to the primers bound to template DNA and adds new primers to the 3’ end of the primer to create a new sequence complementary to the template DNA.
  4. The three steps are repeated 25-30 times to create 2X the amount of DNA each time
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10
Q

Describe the role of primers in PCR:

A
  • Are important since DNA polymerase does not work unless given a section of double-stranded DNA to start on
  • Are specific to a section of DNA (the longer the primer, the less likely that sequence will occur in more than one location)
  • Each primer must be complementary to the opposite strand to the other
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11
Q

Describe the role of restriction endonucleases in cutting DNA:

A
  • Molecular scissors cut DNA molecules into smaller pieces at specific base sequences
  • REs recognise a specific sequence of DNA (called a recognition sequence)
  • Sticky ends (single-stranded overhand): Used when needing to join pieces of DNA together
  • Complementary ends will naturally pair up but must have complementary overhand (eg. Been cut with the same RE)
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12
Q

How is DNA naturally cut in bacteria?

A
  • Occurs as part of their cell defences against foreign DNA (eg. Viruses)
  • Restriction endonucleases act by cutting DNA that doesn’t belong
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13
Q

How do blunt ends of cut DNA (no overhang) differ from sticky ends (single-stranded overhand)?

A

Blunt ends are harder to use for joining pieces of DNA together, but don’t required both samples to be cut with the same RE.

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14
Q

What is the role of DNA ligase in relation to joining DNA?

A
  • Like glue
  • Forms phosphodiester bonds between the deoxyribose sugar and the phosphate in the DNA backbone
  • Helps to create recombinant DNA
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15
Q

What are bacterial plasmids and what is their use?

A
  • Non-essential, extra-chromosomal circular DNA molecule found in bacterial cells
  • May confer additional properties to the bacterial cell (Eg. Antibiotic resistance)
  • Many plasmids used in genetic engineering have been created to have certain useful features including areas with many possible restriction enzyme cut sites
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16
Q

Describe the 3 steps to create recombinant DNA (genetic cloning):

A
  1. Cut the ends of the gene and the ends of the destination DNA (often a plasmid) with the same restriction enzyme
  2. Mix the two pieces of DNA together
    - Molecular movement will mean that with enough time, the ends of DNA will randomly meet
    - Sticky end will hydrogen bond, temporarily holding DNA together
  3. Add DNA ligase
    - Joins backbone together to permanently join the two pieces together
17
Q

What does the term transformations refer to in relation to gene delivery systems?

A

Places by which DNA, taken from one organism, are inserted into another organism using a plasmid (the process by which we place a plasmid into a bacterial cell).

  • Cells are treated to make the cell ‘leaky’ or more fluid for the DNA (cells are ‘competent’)
  • Bacteria are mixed with plasmids and a few cells/ per 1000 will take up the plasmid
18
Q

Describe the process of the selection for transformants:

A
  • To determine if transformation is successful, the ‘transformant’ cells are plated on selective media upon which growth is dependent on the expression of plasmid genes
  • Only cells that take up the plasmid, and express its antibiotic resistance proteins, can thrive off the antibiotic medium = untransformed cells fail to grow
19
Q

Describe the process of gel electrophoresis:

A
  • Used to separate DNA fragments according to length
  • DNA is extracted from cells or obtained using PCR and then cut using restriction enzymes
  • Fragments of DNA are loaded/put into a special gel (agar) that is subjected to an electric current
  • Because DNA fragments are negatively charged, the fragments are pulled towards the positive electrode
  • The shorter, lighter fragments of DNA move more quickly than the larger pieces and so move towards the positive electrode more quickly
  • The DNA then becomes sorted by size
20
Q

Describe the process of gel electrophoresis:

A
  • Used to separate DNA fragments according to length
  • DNA is extracted from cells or obtained using PCR and then cut using restriction enzymes
  • Fragments of DNA are loaded/put into a special gel (agar) that is subjected to an electric current
  • Because DNA fragments are negatively charged, the fragments are pulled towards the positive electrode
  • The shorter, lighter fragments of DNA move more quickly than the larger pieces and so move towards the positive electrode more quickly
  • The DNA then becomes sorted by size
21
Q

What is DNA sequencing?

A

The determination of the actual order of the bases (sequence of nucleotides) in a DNA strand.
-Uses a modified PCR reaction

22
Q

Describe the 6 steps of DNA sequencing:

A
  1. DNA is separated into single-stranded molecules by heating.
  2. The single-stranded DNA is added to 4 separate tubes, each containing:
    - DNA polymerase (Taq)
    - Free nucleotides of all base types (A,T,C,G)
    - Short primers
  3. Each of the four tubes contains a small amount of one dideoxynucleotide: ddA, ddC, ddG, or ddT.
  4. The tubes are cooled and replication is allowed to proceed.
  5. Complementary DNA strands are formed in each tube, according to base pairing rules. Occasionally, a dideoxynucleotide is incorporated into the growing DNA strand and replication stop, where the length of each DNA chain indicates where that particular nucleotide occurred.
  6. At the end of the replication process, each tube contains a number of DNA fragments of different lengths.
    - All these fragments of DNA can be separated using gel electrophoresis
23
Q

List 5 gene technology uses:

A

-Identify the alleles of genes responsible for serious medical conditions
-Identify cancer types to better match drug treatments
-Screening for genetic diseases in individuals or embryos
-Generate large amounts of protein for commercial or medical reasons
• Gene therapy for people with diseases

24
Q

What are transgenic organisms? Give an example:

A

Any organism that possesses a ‘foreign’ gene or segment of ‘foreign’ DNA in their genome as a result of human experimentation.
-Can insert genes either in germline cells (all offspring will carry the gene) or into somatic cells only (offspring unaffected)
Eg. Crops have genes introduced in the germline to:
-Increase pesticide resistance
-Increase yield
-Increase shelf life
-Change tolerance to heat, cold and other growing conditions

25
Q

Describe the evolutionary consequences of genetic screening:

A
  • Decisions which individuals make on the basis of the results of genetic screening have the potential to change the human gene pool
  • A person who knows they are a carrier of a detrimental trait may choose not to have children
  • Parents may choose to terminate a pregnancy if the foetus is shown to have a genetic abnormality or inherited disease
  • Screening prior to implanting embryos (using IVF) leads to few offspring with genetic disorders

Bio Yr12 (21 decks)

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