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8.4 all Flashcards

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1
Q

What is recombinant DNA technology?

A

The combining of different organism’s DNA

scientists alter genes to improve industrial processes and medical treatments.

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2
Q

What is the first step in recombinant DNA technology?

A

To produce or isolate the fragments of DNA to be recombined with another piece of DNA.

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3
Q

What are the three methods to create fragments of DNA?

A
  1. Reverse transcription
  2. Restriction endonucleases
  3. Gene machine
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4
Q

What is the first step of using reverse transcription to create fragments of DNA?

A

Reverse transcriptase makes DNA copies from mRNA.

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5
Q

What happens after reverse transcriptase makes DNA copies from mRNA?

A

A cell that naturally produces the protein of interest is selected.

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6
Q

What happens after a cell that naturally produces the protein of interest is selected?

A

The reverse transcriptase enzyme joins the DNA nucleotides with complementary bases to the mRNA sequence.

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7
Q

What is made when reverse transcriptase joins the DNA nucleotides with complementary bases to the mRNA sequence?

A

Single stranded DNA (cDNA).

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8
Q

What is used to make cDNA double stranded?

A

DNA polymerase.

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9
Q

What is an advantage of using reverse transcriptase to produce DNA fragments?

A

The cDNA is intron free, because it is based on the mRNA template.

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10
Q

What are restriction endonucleases?

A

Enzymes that cut DNA at specific nucleotide sequences.

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11
Q

Where do restriction enzymes naturally occur?

A

In bacteria - as a defense mechanism.

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12
Q

What are recognition sequences?

A

The DNA base sequences that are complementary to the active site of restriction enzymes.

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13
Q

What are the two types of ways that the ends are cut?

A
  1. Some ends are cut in the same location in the double strand, creating a blunt end.
  2. Others are cut to create staggered ends and exposed DNA bases.
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14
Q

What are the exposed staggered ends?

A

Palindromic.

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15
Q

Why are the exposed staggered ends called sticky ends?

A

They have the ability to join DNA with the complementary base pairs.

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16
Q

What is the first step of using a gene machine to create DNA fragments?

A

Scientists examine the protein of interest to identify the amino acid sequence and work out what the mRNA and DNA should be.

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17
Q

What happens after scientists work out what the mRNA and DNA should be?

A

DNA sequence is entered into the computer, which checks for biosafety and biosecurity.

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18
Q

What happens after the computer has checked for biosafety and biosecurity?

A

create small sections of overlapping single strands of nucleotides that make up the gene (oligonucleotides).

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19
Q

What happens after the oligonucleotides are created?

A

They can be joined to create the DNA for the entire gene.

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20
Q

How is PCR used in gene machine?

A

To amplify the quantity of DNA and double the strand.

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21
Q

What is the main advantage of reverse transcriptase?

A

mRNA present in cell is from actively transcribed genes, so lots of the mRNA of interest to make cDNA.

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22
Q

What is an advantage of restriction endonuclease?

A

Sticky ends on DNA fragment make it easier to insert to make recombinant DNA.

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23
Q

What is an advantage of gene machines?

A

Can design exact DNA fragment you want, with sticky ends, labels and preferential codons.

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24
Q

What is a disadvantage of reverse transcriptase?

A

More steps, so more time consuming and technically more difficult.

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25
What is a disadvantage of restriction endonuclease?
Still contains introns.
26
What is a disadvantage of gene machine?
Need to know the sequence of amino acids or bases.
27
In in vivo cloning, where must a promoter region be added?
At the start of the DNA fragment.
28
What is a promoter region?
A sequence of DNA which is the binding site for RNA polymerase to enable transcription to occur.
29
In in vivo cloning, where must a terminator region be added?
At the end of the DNA fragment.
30
What is a terminator region?
Causes RNA polymerase to detach and stop transcription, so only one gene at a time is copied into mRNA.
31
What is a vector (in in vivo cloning)?
Carries the isolated DNA fragment into the host cell.
32
In vivo - what are the most common vectors?
Plasmids.
33
What are plasmids?
Circular DNA, which only contain a few genes.
34
In vivo - how is the plasmid cut open?
Using the EXACT same restriction endonuclease (as was used to cut out DNA).
35
In vivo - why is the same restriction endonuclease used?
So it cuts at the same sequence of bases, creating the same sticky ends.
36
In vivo - why is it important to create the same sticky ends?
So the DNA fragment sticky ends (exposed nucleotides) are complimentary to the sticky ends on the plasmid.
37
In vivo - what happens after the RE cuts open the plasmid?
The DNA fragment and cut plasmid are combined and the enzyme ligase sticks them together (anneals them).
38
In vivo - how does ligase anneal the DNA fragment and cut plasmid?
It catalyses the condensation reaction to form phosphodiester bonds between nucleotides.
39
In vivo - what needs to happen after ligase anneals the DNA fragment and cut plasmid?
The vector (plasmid with recombinant DNA) needs to be inserted into the host cell.
40
In vivo - what is needed for the vector to be inserted into the host cell?
The cell membrane of the host cell must be more permeable.
41
In vivo - how does the cell membrane of the host cell become more permeable?
The host cells are mixed with Ca2+ and heat shocked - enables the vector to enter the host cell's cytoplasm.
42
What 3 issues can occur in in vivo cloning?
1. The recombinant plasmid doesn't get inside the cell. 2. The plasmid re-joins before the DNA fragment enters. 3. The DNA fragment sticks to itself, rather than inserting into the plasmid.
43
What do scientists need to do as a result of the issues that can occur in in vivo cloning?
Must identify which bacteria successfully took up a recombinant plasmid.
44
In vivo cloning - what can be used to identify which bacteria successfully took up a recombinant plasmid?
Marker genes.
45
In vivo cloning - how is the host cell grown?
A fermenter is used to grow multiple copies of the host cell, which have been identified as containing the recombinant plasmid.
46
What is genetic fingerprinting?
The analysis of VNTR DNA fragments -> which can be used to determine genetic relationships and the genetic variability within a population.
47
What are VNTRs?
Introns consist of many variable number tandem repeats (VNTRs).
48
What are the stages of genetic fingerprinting?
1. Collection 2. Extraction 3. Digestion 4. Separation 5. Hybridisation 6. Development 7. Analysis Crazy Elephants Don't Stop Hydrating During Autumn.
49
Genetic fingerprinting - collection & extraction:
The smallest sample of DNA can be collected for genetic fingerprinting. If the sample of DNA is too small, then PCR is used to amplify the amount of DNA.
50
Genetic fingerprinting - digestion:
Restriction endonucleases are added to cut the DNA into smaller fragments. Enzymes which cut close to the target VNTRs are added.
51
Genetic fingerprinting - separation - first step:
The DNA samples are loaded into small wells in agar gel. The gel is placed in a buffer liquid with an electrical voltage applied.
52
Genetic fingerprinting - separation - what happens after the DNA samples are loaded?
The DNA is negatively charged, so the DNA samples move through the gel towards the positive end.
53
Genetic fingerprinting - separation - gel electrophoresis:
The agar gel creates resistance for the moving DNA, and smaller pieces of DNA can move faster + further along the gel.
54
Genetic fingerprinting - hybridisation:
DNA probes are mixed with the single stranded DNA VNTRs on the agar gel for them to bind (hybridise).
55
What are DNA probes?
Short, single-stranded pieces of DNA complementary in base sequence to the VNTRs. The probes are radioactive or fluorescently labelled.
56
Genetic fingerprinting - development:
The agar gel will shrink and crack as it dries, and the VNTRs and DNA probes are transferred to a nylon sheet.
57
Genetic fingerprinting - analysis:
The position of the DNA bands are compared to identify genetic relationships, the presence of a disease-causing gene and to match unknown samples from a crime scene.
58
What are the uses of genetic fingerprinting?
1. Forensic science 2. Medical diagnosis 3. To ensure animals and plants are not closely related before being bred.
59
What needs to happen after the DNA fragments have been isolated?
They need to be cloned to create large quantities, which can be done either in vivo or in vitro.
60
How can fragments be cloned in vitro?
Fragments of DNA can be amplified in vitro via the polymerase chain reaction (PCR), done by an automated machine.
61
First step of the PCR method:
Temperature is increased to 95 degrees to break hydrogen bonds and split the DNA into single strands (denaturing).
62
PCR - what happens after the temperature is increased to 95?
Is then decreased to 55, so that primers can attach (annealing).
63
PCR - what happens after the temperature is decreased to 55?
DNA polymerase then attaches complementary free nucleotides and makes a new strand to align next to each template (synthesis).
64
What are the advantages of PCR?
1. Automated (more efficient) 2. Rapid (100 billion copies of DNA can be made within hours) 3. Doesn't require living cells (quicker and less complex techniques needed).
65
What are DNA probes used for in genetic screening?
To locate specific alleles of genes and to screen patients for heritable conditions, drug responses or health risks.
66
How do DNA probes identify specific alleles?
They are created to have a specific complementary base sequence to the alleles that is being screened for.
67
How is the DNA sample made single stranded?
Is heated, which causes the H bonds between bases to break (denaturing).
68
How do the DNA sample and probes anneal?
Any complementary sequences can align and form hydrogen bonds.
69
Can some of the DNA samples anneal back together?
Yes.
70
What is the first step of locating specific alleles of genes?
The DNA base sequence must be known to then create the DNA probe.
71
Locating specific alleles of genes - what happens after the DNA base sequence is determined?
The fragment of DNA can be produced using a gene machine.
72
Locating specific alleles of genes - what happens after the fragment is amplified?
The label is then added, either a radioactive nucleotide (containing p32 isotopes) or a fluorescent label.
73
Locating specific alleles of genes - what happens after hybridisation?
The DNA is washed so that any unbound DNA probes are washed away.
74
Locating specific alleles of genes - results?
The presence of the radioactive or fluorescent label indicates that the allele of interest is present in the patient's DNA.
75
What is genetic screening?
People have their family history researched to consider the likelihood of them carrying any alleles linked to disease.
76
What is personalised medicine used for?
Certain drugs are more or less effective depending on your genotype.

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