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Topic 8) Control Of Gene Expression > 8B > Flashcards

8B Flashcards

1
Q

What does recombinant DNA technology mean?

A

Recombining DNA by transferring DNA from one organism into another.

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2
Q

How is recombining DNA possible?

A

As genetic code is universal
Transcription & translation similar in most organisms

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3
Q

What is created after recombining DNA?

A

A transgenic organism
Which can produce proteins coded for by original DNA sequence

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4
Q

What are the 3 ways of obtaining a DNA fragment?

A
  1. Reverse transcriptase
  2. Restriction endonuclease
  3. Gene machine
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5
Q

What is reverse transcriptase?

A

Enzyme that makes a DNA copy from an RNA template
Found in RNA retroviruses - e.g. HIV

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6
Q

How is reverse transcriptase used to obtain a DNA fragment?

A
  • mRNA molecule complementary to target gene is isolated from cells
  • mixed with free DNA nucleotides & reverse transcriptase
  • reverse transcriptase uses mRNA as template to synthesise new cDNA strands
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7
Q

Why is it easy to use mRNA to obtain DNA fragments?

A

Most cells only contain 2 copies of each gene
But cells that produce proteins coded coded for by target gene contain many mRNA molecules complementary to target gene
mRNA is found in the cytoplasm

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8
Q

What is meant by cDNA?

A

Complementary DNA

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9
Q

Why will cDNA produced be shorter than original DNA sequence?

A

cDNA won’t contain introns
As it’s complementary to mRNA which doesn’t contain introns

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10
Q

What is restriction endonuclease?

A

Enzyme that recognises specific recognition sequences and cuts DNA at these places

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11
Q

How is restriction endonuclease used to obtain DNA fragments?

A

If recognition sites are present at either side of DNA fragment you want
DNA sample incubated with specific restriction endonuclease
Restriction endonuclease cuts DNA fragment out via condensation reaction
Leaving sticky ends sometimes

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12
Q

What is a recognition sequence?

A

Specific palindromic sequences
Different restriction enzymes cut at different specific recognition sequences
As it has to be complementary to its active site

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13
Q

What are palindromic sequences?

A

Sequence of nucleotides that consist of antiparallel base pairs

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14
Q

What are sticky ends?

A

Small tails of unpaired bases at each end of fragment
Can be used to bind DNA fragment to another that has complementary sequences

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15
Q

How is a gene machine used to obtain DNA fragments?

A

Sequence required is designed
First nucleotide requires is fixed to a support
Next nucleotides then added in process that includes adding protecting groups
Oligonucleotides produced, are broken off from support
Protecting groups removed
Oligonucleotides joined together to make longer DNA fragments

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16
Q

What do protecting groupsdo?

A

Make sure nucleotides join at right points
Prevent unwanted branching

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17
Q

What are oligonucleotides?

A

Short sections of DNA, around 20 nucleotides long

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18
Q

What is a benefit of using a gene machine?

A

Pre-existing DNA template not needed
Instead, database contains information to produce DNA fragments
So DNA fragments doesn’t need to exist naturally

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19
Q

What are the 2 types of gene cloning?

A

In vitro cloning
In vivo cloning

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20
Q

What is ** in vitro cloning**?

A

Where copies of DNA fragments are made outside of an organism
Using polymerase chain reaction (PCR)

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21
Q

Outline in vitro cloning:

A
  1. Reaction mixture set up, containing: DNA sample, free nucleotides, primers, DNA polymerase.
  2. DNA mixture heated to 95C to break hydrogen bonds between the double DNA strand.
  3. Mixture cooled to 55C, allowing primers to anneal to strands.
  4. Heated to 72C, so DNA polymerase can work to join adjacent nucleotides.
    Complementary strands formed due to specific base pairing.
  5. 2 new copies of DNA fragment formed.
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22
Q

What are primers?

A

Short single-stranded sequence of nucleotides
Complementary to bases at start of target DNA fragment
So induces start of cloning

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23
Q

What is a thermocycler?

A

Computer controlled machine
Varies temperature
Used in in vitro cloning

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24
Q

What is *in vivo cloning?

A

Copies of DNA fragments produced in a living organism
Using a vector

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25
What is a **vector**?
Something used to **transfer DNA** into a **host cell** E.g. plasmids, bacteriophage
26
What are the **3 stages** of **in vivo cloning**?
1. Making **recombinant DNA** 2. **Transforming** cells 3. **Identifying** transformed cells
27
Outline **making recombinant DNA** in **in vivo** cloning:
1. Isolate **vector DNA** & cut it open using same **restriction endonuclease** used to obtain DNA fragments - so that **sticky ends** of fragment complementary to those of vector DNA. 2. **Ligation**: vector DNA & DNA fragment mixed with enzyme **DNA ligase**, which **joins sticky ends** 3. New combination of DNA bases = **recombinant DNA**
28
What is **DNA ligase**?
**Enzyme** used to **stick DNA fragment & vector DNA** together.
29
Outline **transforming cells** in **in vivo** cloning:
If **plasmid vector** is used: - host bacterial cells placed in ice-cold solution to make cell walls more permeable - **heat-shocked**, encouraging them to uptake plasmids If **bacteriophage vector** is used: - it will infect host bacterium by **injecting** its DNA into it
30
What is a **transformed cell**?
**Host** cells that take up vector containing **target gene**
31
Outline **identifying transformed cells** in **in vivo** cloning:
1. **Marker genes** inserted into vectors during making recombinant DNA - so transformed host cells also will contain marker gene 2. Host cells grown on agar plate, divide & replicate, creating a colony of cloned cells - transformed cells would produce colony of cloned cells with marker gene 3. Marker gene would be for: - **antibiotic resistance**: cells placed on plate with specific antibiotic, so only transformed cells survive - **florescence**: scientists can identify under UV light 4. Identified transformed cells allowed to grow more, producing lots of copies of cloned genes
32
How do we **produce proteins** from transformed cells?
Make sure vector contains specific **promoter** and **terminator** regions.
33
What is a **promoter region**?
Region that tells **DNA polymerase** where to **start transcribing** from
34
What is a **terminator region**?
Region that tells **RNA polymerase** where to **stop transcribing**
35
What is a **benefit** of **recombinant DNA technology** in **agriculture**?
Crops transformed to give higher yields, be more nutritious, have resistance to pests & droughts.
36
What is a **concern** of **recombinant DNA technology** in **agriculture**?
Monocultures could become more common, reducing biodiversity. ‘Superweeds’ could occur if transformed crops interbreed with wild plants.
37
What is a **benefit** of **recombinant DNA technology** in **industry**?
Using enzymes produced by transformed organisms in industrial processes in large quantities at low costs.
38
What is a **concern** of **recombinant DNA technology** in **industry**?
People believe genetically engineered organisms could introduce toxins into the food industry Smaller companies without access to biotechnology could loose business
39
What is a **benefit** of **recombinant DNA technology** in **medicine**?
Drugs & vaccines produced by transformed organisms - quickly, cheaply & in large quantities.
40
What is a **concern** of **recombinant DNA technology** in **medicine**?
Technology could be used unethically e.g. to make designer babies.
41
What is the **human genome**?
**Entire set of DNA**, including the genes, of an organism. Around 3 billion bases long
42
What are the **uses** in knowing the sequence of the **human genome**?
Genetic testing Trace migration patterns Gene therapy treatments Monitor mutations Development of vaccines
43
What are some of the **benefits** of **genetic screening**?
Enables people to make sensible **lifestyle choices** if they’re at risk of developing a disease Enables **potential parents** to chose whether having their own **biological children** could risk passing on a **harmful allele** Enables people to participate in **research & clinical trials** to develop our understanding of **genetic disorders**
44
What are some **concerns** of **genetic screening**?
Could lead to depression/anxiety for someone to find out they could develop an incurable, non-preventable disease. Genetic discrimination e.g. paying higher life insurance Could cause parents to abort foetuses without desired genetics
45
What is a **DNA probe**?
**Single-stranded** sequences of **DNA** (or RNA) used to search for its **complementary sequence** in **sample genome**. Also known as a **gene probe**
46
What are the main **uses** of **gene probes**?
Help identify inherited conditions Determining how a patient will respond to specific drugs Identifying health risks
47
How are **gene probes** created?
Order of nucleotides on **mutated** genes is determined by **DNA sequencing** **DNA fragment** with **complementary bases** to **mutated** portion of gene is produced DNA probe formed by **radioactively or fluorescently labelling** this DNA fragment
48
How do we produce **multiple copies** of **DNA probe**?
Using **PCR** techniques (**in vitro** cloning)
49
How does **gene probe** detect presence of **mutated gene**?
Probe added to **single-stranded DNA fragments** from person being screened If person has mutated gene, some DNA fragments will have **complementary nucleotide sequence** to **probe**, so probe will bind to complementary bases on DNA These **DNA fragments** now **labelled** with **probe**
50
What would happen if **mutated gene** is **present** when tested by a **DNA probe**?
**DNA probe** will have been taken up Probe would be **florescent** or **radioactive**, so scientists can use **UV** or **X rays** to detect for **probe** and therefore presence of **mutated gene**
51
What would happen if **mutated gene** is **not** present when tested for by a **DNA probe**?
**DNA probe** will not have been taken up So no florescence or radioactivity
52
What is a **DNA microarray**?
Glass slide with microscopic spots of **several different DNA probes** attached to spots Allows for screening of several different genes at the same time
53
What is **gene therapy**?
When **recombinant DNA** is used to treat to treat human diseases
54
How can we treat diseased caused by **2 mutated recessive alleles**?
Can add a **working dominant allele** so it’s expressed instead By inserting a DNA fragment into original DNA using a vector
55
How can we treat diseases caused by a **mutated dominant allele**?
Can ‘silence’ gene by **sticking DNA in the middle** of it, so it **no longer works** By inserting a DNA fragment into original DNA using a vector
56
What is **genetic fingerprinting**?
A diagnostic tool Used in forensic science, medical diagnosis, plant & animal breeding Based on fact that DNA of every individual is unique
57
What is **gel electrophoresis**?
Process involved in **DNA fingerprinting** To create DNA ladder
58
Outline **gel electrophoresis**:
Slab is prepared - usually agarose gel Samples containing **DNA fragments** loaded into wells **Electrical current** passed through gel As **DNA fragments** are **negatively charged**, they move towards **positively charged end** Smaller fragments move quicker than larger, so will have travelled further by the time current is stopped Fragments labelled with dye/ radioactivity so can be seen
59
What does **VNTRs** stand for?
Variable number tandem repeats
60
What are **VNTRs**?
**Base sequences** in genome that **don’t code** for anything, Just **repeat** themselves over & over again
61
How are **VNTRs** used in **genetic fingerprinting**?
Number of times these sequences repeat varies between individuals, so length of sequences in nucleotides differs Also, number of times VNTRs appear in different places differs So they can be compared between individuals in genetic fingerprinting
62
Outline the process of **genetic fingerprinting**
1. DNA extracted from sample 2. DNA is digested using **restriction endonucleases** which cut near **VNTRs** to create **fragments** 3. **Florescent tags** added to all DNA fragments 4. Sample undergoes **PCR** to create many copies of fragments 5. Fragments separated using **gel electrophoresis** 6. Gel placed under **UV light** so DNA can be seen as **bands**
63
What if scientists aren’t interested in all fragments?
**Probes** added after **gel electrophoresis**
64
How are **probes** added after gel electrophoresis?
Gel immersed in **alkali** to **separate** double **strands** into single strands DNA fragments transferred to **nylon** membrane **Radioactive** or **florescent probes** added, bind to **target DNA** sequences in **VNTRs** **UV light/X rays** used to see bands
65
What is the purpose of a **DNA ladder*?
Runs alongside samples being tested Helps **determine length** of fragments in sample
66
How many **different locations** in the human genome can **VNTRs** be found?
Over 1,000
67
How can DNA for genetic fingerprinting be obtained?
Mouth swabs Remains of blood, hair & skin Inside bones
68
What is special about the way **restriction endonucleases** work in **genetic fingerprinting**?
Digest DNA at sites just before & after **VNTRs**, leaving VNTRs intact
69
What kind of **banding pattern** would suggest a close genetic relationship?
If around **half** the bands match.

Topic 8) Control Of Gene Expression (1 decks)

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