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Recombinant DNA technology! Flashcards

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1
Q

Define recombinant DNA

A

DNA that has been formed artificially by combining two or more fragments from different sources

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2
Q

What method would you use to amplify a gene of interest?

A

PCR

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3
Q

Applications of genetic manipulation?

A
  • medicine and testing
  • Pharmaceutically or commercially important protein can be expressed in a host organism
  • DNA profiling in forensic science and paternity testing.
  • Genetic manipulation of organisms
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4
Q

What do type II restriction enzymes do?

A

recognise, and cleave
at, specific DNA sequences.
* 4 or 6 base-pairs (bp) in length
* “sticky” or blunt ends.
* Cutting sites are palindromic

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5
Q

What enzyme is used to break into a plasmid?

A

Linearase

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6
Q

What is EcoRI and what does it do?

A

A restriction enzyme
Recognises a 6 bp target site and cleaves DNA to leave ‘sticky ends’
- breaks phosphodiester bond

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7
Q

What is HaeIII and what does it do?

A

A restriction enzyme
Recognises a 4 bp target site and cleaves DNA to leave blunt ends
- can’t stick back together

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8
Q

What do restriction enzymes leave at the 3’ and 5’ ends?

A

OH at 3’ end
Phosphate at 5’ end

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9
Q

In DNA analysis, what does DNA ligase do?

A

Forms covalent bonds between two DNA fragments with the same sticky end
It can also join blunt ended fragments, but with less efficiency than sticky ended fragments
- needs ATP to reform phosphiodiester bonds

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10
Q

What 2 enzyme steps are involved in inserting a fragment of DNA into a plasmid?

A

DIGESTING with restriction enzyme e.g. EcoRI
Ligating with DNA ligase

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11
Q

What 3 requirements must plasmids used as vectors in gene cloning have?

A

One or more unique restriction enzyme sites.
An origin of replication specific to the host organism.
A selectable marker (usually an antibiotic resistance gene)

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12
Q

What do isochizomers do?

A

Recognise the same site and cut it in the same way

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13
Q

What do neoschizomers do?

A

Recognise the same site but cut it in a different way to each other

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14
Q

Define transgenic animal

A

An animal that has exogenous DNA inserted into its genome

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15
Q

What kind of bonds does DNA ligase form in DNA?

A

Covalent phosphodiester bonds

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16
Q

Between which kind of ends does DNA ligase form bonds at a higher efficiency, sticky or blunt?

A

Sticky

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17
Q

Define vector

A

A DNA molecule that carries the exogenous
DNA fragment into a host cell

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18
Q

What are the most commonly used vectors in recombinant DNA technology?

A

Modified plasmids

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19
Q

What are some desirable but not essential features of a plasmid vector?

A

High copy number
Small size (a few thousand bp)
A means of distinguishing recombinant plasmids (i.e. with exogenous DNA inserted) from non-recombinant plasmids

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20
Q

What us pUC18?

A

A typical cloning vector

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21
Q

What is transformation of E. Coli cells achieved by?

A

Heat shock (42°C): Temporarily opens pores in the membrane and uses Ca2+ ions

Electroporation: Electric pulse to allow DNA entry.

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22
Q

What is blue-white selection in LacZ?

A

Cleaved by b-galactosidase
- recombinant plasmid colonies = white
- non-recombinant colonies = blue
LacZ distinguishes the colonies

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23
Q

What is a gene library?

A

large collections of cloned DNA fragments representing all or most of genes present from an organism

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24
Q

What are the two types of clones?

A
  1. Genomic clones
  2. cDNA clones
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25
What is cDNA and why would it be used?
Cloning of cDNA (DNA complementary to mRNA) generates cDNA clones. * Used if the protein made by the gene is to be produced by recombinant bacteria
26
Why would you use genomic clones over cDNA clones?
Used if the promoter or the intron-exon structure is to be analysed
27
What is cDNA?
Synthesized from mRNA using reverse transcriptase. Contains only exons (no introns). Represents only expressed genes (transcriptome)
28
How is cDNA created?
Isolate mRNA (often using poly-A tail and oligo(dT) beads). Use reverse transcriptase to synthesize single-stranded cDNA. Add a primer (e.g., random hexamers or oligo-dT). Use DNA polymerase (e.g., DNA polymerase I) to synthesize the second strand.
29
What are the uses of PCR?
- cloning genes - measuring gene expression - DNA profiling - diagnosis of genetic and pathogen induced disease e
30
What 6 things do you need to perform PCR?
Target DNA Primers – 2 short segments of single stranded DNA Taq DNA polymerase heat stable, from Thermus aquaticus dNTPs Buffer containing Mg2+ Thermal cycler
31
Explain what each primer used in PCR corresponds to
The “Forward” primer has the same sequence as a stretch of the 5′ end of the top strand of the DNA fragment to be amplified. The “Reverse” primer corresponds to the 5′ end of the bottom strand of the DNA fragment to be amplified
32
What are the 3 steps of the PCR cycle?
Denaturation - 95°C (30-60s) denature DNA into single strands Annealing - 40-65°C (30s) allow primers to anneal to ssDNA Extension - 72°C (1-3 min) DNA synthesis
33
How much DNA do you get per PCR cycle?
The amount of target DNA doubles during each cycle of PCR
34
What is the role of Taq DNA polymerase in PCR?
Synthesizes new DNA strands.
35
Why not use normal DNA polymerase in PCR?
because it would denature (break down) at the high temperatures required during the denaturation step of the PCR cycle
36
Why do you need proof reading polymerases in PCR?
Taq is error prone, 285/10^6 bases mis-incorporated
37
What are proof-reading polymerases?
RNAase H-like domains - 3' to 5' proofreading - have a longer half life
38
What are Hot Start Enzymes?
DNA polymerases designed to be inactive at room temperature, preventing unwanted reactions during PCR setup, and then activated at a higher temperature during the PCR cycle
39
What is agarose?
A polysaccharide from seaweed
40
What does agarose gel electrophoresis do?
Separates DNA and RNA molecules according to their size
41
In gel electrophoresis, which electrode do DNA and RNA migrate towards?
Positive
42
What is the rate of migration in gel electrophoresis inversely proportional to?
Rate of migration of linear molecules is inversely proportional to the log10 of their size
43
When expressing eukaryotic genes in E. Coli, how would you overcome the fact that bacterial cells cannot remove introns?
Obtain eukaryotic gene from a cDNA library
44
When expressing eukaryotic genes in E. Coli, how would you overcome the fact that eukaryotic promoters and terminators do not function in prokaryotic cells?
Use expression vector that contains a prokaryotic promoter and terminator
45
When expressing eukaryotic genes in E. Coli, how would you overcome the fact that eukaryotic mRNAs do not have ribosome binding sites?
Use an expression vector containing a ribosome binding site immediately before the site of cDNA insertion.
46
What is an expression vector?
a specially designed plasmid (or viral vector) used to introduce a gene into a host cell and ensure it is expressed (i.e., transcribed and translated into protein)
47
Some important proteins made by GM organisms
48
What is the pGEM3Z plasmid used for?
It is a plasmid vector used to clone a gene and transcribe it in vitro to produce RNA using T7 or SP6 promoters.
49
What is produced during in vitro transcription using SP6 polymerase?
RNA transcripts of the inserted gene
50
What is the pure spike protein mRNA packaged into to create covid vaccine?
Lipid nanoparticles, using pseudouridine to transcribe in vitro
51
What are common uses of in vitro transcribed RNAs?
RNA vaccines, RNA probes, RNA structure-function studies, and translation assays
52
How can tandem repeats be used in forensic analysis?
Number of repeats varies between individuals → genetic fingerprint
53
Describe the process of using tandem repeats to identify individuals
Extract DNA from sample (blood, hair, etc.) Use PCR to amplify specific STR regions (e.g. at 13–20 loci). Separate PCR products by capillary electrophoresis. Compare the lengths of STRs → build a DNA profile
54
What 3 different blotting techniques are used to analyse different nucleic acids?
55
Blotting mnemonic
SNoW DRoP Southern → DNA Northern → RNA Western → Protein
56
what is qPCR?
Quantitative PCR - detects presence of DNA
57
What is qRT-PCR?
Quantitative reverse transcriptioin PCR Detects and quantifies RNA in real time Fluorescent dye or probe (e.g. TaqMan) emits signal as DNA is amplified
58
how are qPCR and qtPCR monitored?
fluorescent dye (SYBR) or a sequence specific DNA probe (TaqMan probe)
59
What is the Ct value?
The number of cycles required to reach a defined threshold quantity of product - can determine copy number The Ct value (cycle threshold) reflects how much virus is present: Low Ct = high viral load High Ct = low viral load
60
Which three genes does qRT-PCR covid 19 testing test for?
S, N and ORF1ab
61
What does more target sequence at the start of PCR mean?
fewer cycles needed to reach threshold
62
What is a transgenic animal?
A transgenic animal is one that has exogenous DNA inserted into its genome.
63
What are transgenic animals used for?
They are used to improve farm animals, produce valuable proteins in milk, and for scientific research.
64
How are transgenic animals made?
By pronuclear microinjection: DNA is injected into the male pronucleus of a fertilised egg.
65
What percentage of offspring are transgenic using microinjection?
Approximately 25% are transgenic.
66
What is AquAdvantage salmon?
A genetically modified salmon that grows faster due to a growth hormone gene, approved in the US and Canada. - uses antifreeze protein
67
What is pharming?
The production of pharmaceutical proteins in the milk of transgenic animals.
68
How were transgenic goats made?
Took promoter from b-casein goat milk and put it in front of human antithrombin gene - makes protein in milk
69
What is a transgenic plant?
A plant that has exogenous DNA inserted into its genome.
70
What are transgenic plants used for?
To produce useful proteins and improve agricultural traits such as pest resistance and nutrition.
71
What is the Ti plasmid?
A plasmid from Agrobacterium tumefaciens used to transfer DNA into plant genomes.
72
What are the T and Vir regions of the Ti plasmid?
T-region integrates DNA into the plant genome, promotes tumour formation ; Vir-region - virulence region helps transfer the T-DNA.
73
What is Bt corn?
A genetically modified corn that expresses a bacterial insecticidal protein from Bacillus thuringiensis.
74
What is glyphosate-resistant soy?
A transgenic soybean that expresses a bacterial EPSPS enzyme unaffected by glyphosate herbicide.
75
Name three concerns about transgenic plants.
Gene transfer to weeds, unexpected effects on human health, and overuse of herbicides causing resistance.
76
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats. - its like restriction enzymes but with a memory, can find sequence again
77
What is Cas9?
An endonuclease enzyme that cuts DNA at specific sites guided by RNA.
78
Where was CRISPR/Cas9 discovered?
In bacteria and archaea, as part of their adaptive immune systems. - derived from bacteriophage infecting a prokaryote
79
What are crRNA and tracrRNA?
They form a complex that guides Cas9 to the target DNA for cleavage.
80
What is a sgRNA?
A single guide RNA combining crRNA and tracrRNA used to direct Cas9 in genome editing.
81
What happens after Cas9 cuts DNA?
The break is repaired either by NHEJ (error-prone) or HDR (precise).
82
Two double strand breakage repairs in eukaryotes?
Homology directed repairs (HDR) Non-homologous end joining (NHEJ)
83
What is NHEJ?
Non-Homologous End Joining: a quick, error-prone repair process that can knock out gene function. - repairs double stranded breaks in any phase of cell cycle
84
What is HDR?
Homology-Directed Repair: a precise repair mechanism using a DNA template for gene correction or insertion. - only repairs double stranded breaks in S-phase and early G2 phase
85
Name three organisms where CRISPR has been used.
Humans, mice, and zebrafish (also yeast, rice, tobacco, etc.).
86
How was CRISPR used in yeast?
The ADE2 gene was deleted and function was restored by adding the gene from another species.
87
What is CAR T-cell therapy?
A cancer treatment where T-cells are genetically modified using CRISPR to better target tumors. - also used in gene therapy
88
Summarise the difference between NHEJ and HDR.
NHEJ is error-prone and used for gene knockouts; HDR is precise and used for editing or replacing genes.
89
Summarise the applications of transgenic and CRISPR technologies.
Used for research, agriculture, pharmaceuticals, and gene therapy by modifying genomes in plants, animals, and humans.
90
What is CRISPR?
It’s a natural immune system in bacteria and archaea that protects them from viruses (bacteriophages)
91
Explain how CRISPR is used in gene modification
Design a guide RNA (gRNA or sgRNA): Combines crRNA and tracrRNA into a single strand. Matches the target DNA sequence. Cas9 + sgRNA introduced into cells: This complex finds and binds to the matching DNA sequence. Cas9 cuts the DNA: A double-strand break (DSB) is created at the target site. Cells repair the break using one of two pathways: 1. NHEJ – Non-Homologous End Joining Fast, error-prone. 2. HDR – Homology-Directed Repair Accurate, requires a DNA template.

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