Gene cloning and DNA technology Flashcards
1
Q
Whats the definition of gene cloning?
A
A production of multiple identical copies of a DNA fragment
2
Q
What were the 3 phases for the discovery of genetics and the years?
A
1st: 1900s Mendelian theory of hereditary
2nd: 1950-60s Genetic code cracked
- discovered that DNA is the genetic material
- transcription and translation
3rd: 1970s recombinant DNA
3
Q
Whats a Genome?
A
The length of DNA in 1 haploid set of chromosomes (the entire gene set)
4
Q
What are the key steps to gene cloning?
A
- The gene is isolated (DNA)
- Inserted into a vector- recombinant DNA
- transported into host cell
- multiplication of recombinant DNA
- division of hose cell colony/ clones
5
Q
In gene cloning, whats commonly used a the host cell and why?
A
A laboratory strain of E. coli as its benign and easy to grow
6
Q
Whats a common vector to use and why is it so useful?
A
Plasmid vectors
- generate large amounts of DNA
- Replicate independently of the host cell
7
Q
Where are plasmids derived from?
A
Small circular DNAs in bacteria
8
Q
What are plasmids?
A
A circular loop of double stranded DNA
9
Q
Label this bacterium…
A
10
Q
What is the importance of gene cloning?
A
- to generate large amounts of DNA
- To generate a large amount of protein from your gene of interest
11
Q
Why is generating large amounts of proteins from gene cloning good?
A
- gene sequencing and expression
- detecting mutations that cause genetic diseases and determininig susceptibility to disease
- develop new ways to treat disease
- to cure inherited diseases using gene therapy
12
Q
Why is known ing about gene sequencing and expression good?
A
- know about the genes function and regulation in biological processes
- mutagenesis experiments
- understanding the causes of disease e.g. CF
13
Q
When is detecting mutations and susceptibility to diseases commonly used? and for what?
A
- Neonatal/ prenatal screening
Carried out for the occurence of common genetic disorders e.g. CF
- Presymptomatic testing for late onset genetic diseases
Such as; Familial colon cancer, Huntington’s diseases, inhertied breast and ovarian cancer
14
Q
What disease has found a new way to be treated due to gene cloning and how did the treatment compare previously to now?
A
Diabetes
- caused by the production of an insufficient amount of insulin
- previosly treated with insulin made from the pancreas of cows and pigs
15
Q
What are two methods for isolating specific genes?
A
- PCR (polymerase chain reaction)
- Southern blotting and hybridisation
16
Q
When was PCR invented and by who?
A
Invented by Kary Mullis in 1983
17
Q
What does PCR require?
A
- dsDNA template
- pair of oligonucleotide polymers
- DNA polymerase
- Nucleotides
- Primers (that match with either one of the ends of the gene sequence so it only replicates the required gene)
18
Q
What are the steps to PCR? (including the temperatures required at each step)
A
- Denature the template DNA to ssDNA with heat (95˚c)
- Lower temperature to allow primers to anneal (55˚c)
- DNA polymerase extends primer (72˚c)
- go to step one
19
Q
One round of PCR produces how much DNA?
A
DNA is doubled in PCR
20
Q
How fast can PCR amplify up a single moleucle of DNA into a visible amount of DNA
A
approximately 2 hours
21
Q
What can happen to the PCR product?
A
- digested
- sequencing
- cloned
22
Q
Whats the recommended number of PCR cycles to go through and why?
A
Around 35, as any more than that has little positive effect
As a plateau occurs because
- the reagents have depleted
- the polymerase is damaged
23
Q
When the method of PCR was first created, the polymerases were not heat stable and had to be replenished every cycle.
in 1988, what was used instead and why was it so good?
A
Taq DNA polymerase
- optimum temperature of 72˚c
- heat stable at 94˚c
- wont denature at high temperatures
24
Q
How is PCR used to isolate a gene?
A
- Design 2 primers to anneal to either end of the gene
- Generate enough DNA for sequencing and to detect mutations
- Generate enough DNA to digest and clone into an expression vector to make a protein
25
What are the limitations to PCR?
* difficult to amplify the product longer than 10-15 kb
* need to know the sequence flanking your gene
26
Whats southern blotting and hybridisations steps?
* Digest the DNA into fragments using a restriction enzyme such as **EcoRI**
* Run the digest on an agarose gel
* Denature the DNA (usually whilst its still on the gel)
* Transfer the denatured DNA to the membrane (nitrocellulose)
* probe the membrane with labelled ssDNA (aka. hybridisation). its usually done with 32P labelled ATP
* visualise the radioactively tageted sequence
* cna fix the ssDNA to the filter by baking at 80˚c for 2 hours
27
What is Hybridisation used for?
* to identify the fragment containing the gene of interest
* uses the ability of ssDNA to bind to its complementary strand
28
In hybridisation, what does the number of H bonds indicate?
The stability of the DNA duplex
29
Tm stands for melting temperature. Tell me about the Tm of the following...
1. High GC content
2. High AT content
1. High GC content= High Tm
2. High AT content= Low Tm
30
What are the basic engineering tools for constructing and cloning genes? and why they are used
* Plasmid vectors (to put DNA into)
* Restriction enzymes (to cut DNA)
* Gel electrophoresis (to seperate DNA fragments)
* DNA ligase (to join DNA fragments)
31
What size do plasmid vectors need to be and why?
Less than 10,000 bp
because...
* easily purified
* avoid DNA breakdown
32
Give an example of a plasmid vector and what each aspect of its name stands for
**pBR322**
p= plasmid
BR= Bolivar and Rodriquez (the people who discovered it)
322= identifies it
33
What do all of the letters around the plasmid represent?
ampr = Ampicillin resistance gene
tetr = tetracyclin resistance gene
Ori = the origin of replication
EcoRI/ BamHI/ SalI = the restriction enzyme sites
34
If a restriction enzyme site is found within a resistance gene, why can this be useful?
It allows you to test if the gene of interest has gone into the site
35
What are the roles of Restriction enzymes in cells?
* in bacteria to protect them from viruses
* recognise a specific sequence of bases in the DNA
* Bind to the specific sites in DNA
* cut the dsDNA
36
How do restriction enzymes cut the dsDNA?
By hydrolysing the phosphodiester bonds
37
When were restriction enzyems discovered and by who?
In the 1960s by Werner Arber, Hamilton Smith and Daniel Nathans
38
Whats **Host- controlled restriction?**
When some bacterial cells are immune to bacteriophage infection
39
Give an example of a restriction enzyme thats found in *Escherichia coli bacteria* and the role of this enzyme?
**Eco RI Methylase**
This enzyme recognises the double stranded sequence GAATTC and causes specific methylation on A-3 on both strands. This protects the DNA from cleavage by the ECO RI endonuclease
40
Whats DNA methylation?
When DNA is replicated through semi-conservative replication
DNA methylase selectively methylates Hemi-methylated DNA
41
What are the types of restriction enzymes and what are their main functions?
**Exonucleases:** Removing nucleotides from the ends of the DNA strand
**Endonucleases:** Break nucleic acid chains in the interior
42
What are the types of restriction endonucleases and what locations do they cut?
Which one is the preferred restriction enzyme to use?
**Type I:** Cuts DNA 1000 bases from the recognition site
**Type II:** Cuts DNA at the recognition site
**Type III:** Cuts DNA 25 bases from the recognition site
**Type II** is the preferred endonuclease as its useful for recombinant DNA production and cuts and a specific site
43
What are the 2 types of ends that the Type II restriction endonucleases create when they cut?
Can cut and create either Blunt or sticky ends
44
What are blunt and sticky ends and which one is more useful for recombinant DNA formation and when the specific gene is inserted into the vector?
**Blunt ends** are when the enzyme cuts down the middle of the sequence
**Sticky ends** are when the enzymes cut near the end of chains and leave a sticky/ cohesive end that is unpaired.
Sticky ends are more useful because the overhanding sticky end means that ssDNA is more likely to bind to this, which allows the formation of recombinant DNA to be easier
45
Why is gel electrophoresis useful to use for analysing the results of gene cloning?
It allows one to know where the DNA has been cut correctly
46
What is used to visulaise the gel electrophoresis resultd?
Ethidium bromide and UV light
47
Whats the process of ligation catalysed by?
DNA ligase
48
In order to insert the specific gene into the vector what are the conditions needed to do this?
DNA ligase and ATP
49
What is the role of DNA ligase?
To catalyse the formation of phosphodiester bonds between adjacent bases (these bases are initiatially held together by H bonds before ligase come along and forms the phosphodiester bonds)
This process helps to form recombinant DNA
50
What form of ligase do most experiments use?
**T4 DNA ligase**
This is isolated from Bacteriophage T4
51
Why does the process of ligation require ATP?
In order for the enzyme to carry out further reactions the AMP in the enzyme's active site must be replenished by ATP
52
What are the major steps in gene cloning?
1. construction of recombinant DNA molecules
2. Introduce gene and vehicle to host cell
3. selection
4. grow host cells
53
What the options for cloning into a cloning vehicle?
1. Cut vector and gene with same restriction enzymes
2. Cut vector and gene with different restriction enzymes
3. Gene and vector do not contain compativle enzymes
4. Using linkers/ adaptors
5. TA cloning
54
For option 2: Cut vector and gene with different restriction enzymes
What has to be done and why?
have to cut piece of vector out as sticky ends have 2 different sequences as 2 different enzymes were used. Then gene piece can then be added to plasmid
55
For option 3: Gene and vectoe do not contain compatible enzymes, what are the 2 ways that this process can occur?
1. using nucleases and create blunt ends by cutting off the sticky ends
2. Using DNA pol to fill in the gaps at the ends of the chain in order to remove the sticky ends and create a blunt end
56
Tell me what happens with option 4: Using linkers/ adaptors
stick a piece of DNA on the end of gene of interest before reaction with plasmid. DNA will have RE site that’s needed. So when RE added to mix the DNA will be cut which will create a sticky end that compatible with vector.
Or can do it so DNA with already made sticky end is just attached to the gene of interest (cut off phosphate group so no Phosphodiester bond is made. Use polynucleotide kinase after for use as probes or for subsequence PCR
57
For option 5: TA cloning- a method to clone PCR products, whats often used and why?
**Terminal transferase**
This can be used in PCR as a template for a primer, as it catalyses the addition of nucleotides to the 3' terminus of a DNA moleucles.
(unlike DNA polymerases' it does not require a template)
58
When DNA is introduced into living cells it is known as a **transformation**, what are the steps to transformation to ensure it occurs correctly?
1. Prepare competent bacterial cells
* limited normal uptake
* chemical/ physical treatment 2. Introduce DNA into cells
* Select those cells that have taken up the recombinant DNA
59
How is the competent E.coli cells (K12) prepared?
60
Why is transformation an inefficient process?
* Only a small % of plasmids are taken up
* Approximately 0.01% of cells take up DNA
61
In the major steps of gene cloning, one of the steps was selection, why is this done?
To determine which cells harbour the recombinant DNA molecule. This is usually done by the fact that most vectors confer antibiotic resistance.
62
In the pBR322 gene, ampr can be the selectable marker gene, when this is transformed it is placed in a petri dish conraining what?
an agar and ampicillin plate
63
Whats the procedue for the selection of transformed cells?
1. Cut gene + cut vector (pBR322) + ligase + ATP
2. transform into bacteria under the condition: 14˚c O/N, CaCl2 and heat shock
3. grow on agar and ampicillin plate at 37˚c
64
What are the ways that you are able to distinguish bacteria that have taken up recombinant DNA or are just self-ligated vectors?
1. Replica plating (pBR322) vectors
2. Blue/ white selection (pUC vectors)
3. Restriction mapping (any vector)
65
For replica plating (pBR322) what is used as the selectable marker?
How is it transformed?
What does the marker help to distinguish?
tetr is a selectable marker
The bacteria is transformed by growing on agar + ampicillin plate (only the transformed bacteria will grow)
The selectable marker helps us distinguish between recombinant and Self-ligated vectors
66
After the self-ligated vector and recombinant vector (in replica plating) its transferred to a agar + ampicillin plate, what is it then transferred to?
An agar + tetracyclin plate in order to see if its self-ligated or recombinant.
As in a recombinant vector a gene is added between the tetr which inactivates it so tetr wouldnt grow a colony. which means that if a colony grow in the tetracyclin plate, then it must be a self-ligated vector
67
What does the Lac Z gene encode for?
The enzyme Beta galactosidase which converys lactose into glucose and galactose
68
Recombinant DNA can be identified using blue/ white selection. What is X-Gal and how does it help to identify recombinant?
X-Gal is an analog of lactose and turns blue when cleaved by beta galactosidase
69
What are the steps to restriction mapping?
1. transform ligation mix into bacteria
2. plat out on agar + ampicillin
3. Grow up bacteria and extract DNA
4. use restriction enzymes to distinguish between recombinant and self-ligated vectors
70
In order to produce a protein from a cloned gene, whats required?
An **expression vector**
71
What do expression vectors provide?
All the sequences required for the transcription and translation of your gene
72
what is trasncription catalysed by?
RNA polymerase
73
What does RNA Pol bind to on the gene?
The promotor region
74
To ensure high levels of transcription of foreign gene in bacterial cells, what do they need to supply our gene with?
A bacterial promotor
75
Whats usually used as the bacterial promotor?
The lac operon
76
In bacteria, what do the ribosome attach to for translation?
The mRNA via a ribosome binding site
77
In eukaryotes, what does the ribosome bind to for translation?
The 5' end and then scans the mRNA for the first initiation codon
78
What are the choices of a host cell?
1. Bacteria
2. Animal cells
79
What are the positives of using bacteria as a host cell?
* grow in liquid media
* divide every 20 minutes
* do not glycosylate proteins
80
What are the negatives of using animal cells as host cells?
* need to grow attached to a solid matrix
* divide every 18 hours
81
How are animal cells cloned?
Animal cells in a monolayer culture in a multiplate vessel
82
Whats the main function of the **VIII factor** in animal cells?
it plays a vital role in blood clotting
83
What is the commonest form of haemophilia resulted from?
An inability to synthesise the VIII factor
84
What **was** the VIII factor purified from?
human blood
85
Tell me some characteristics of factor VIII?
* very large- 186 kb
* Contains 17 disulphide bridges
* only active after extensive glycosylation
* SV40 (simian virus 40) promotor
86
What are genetically modified animals useful for?
* producing **pharmaceuticals** for animals and humans
* A source of **cells, tissues and organs** that closely match humans in order to be transplanted with a lower risk of rejection
* produce **high value materials** (surgical sutures)
* produce highly specific **antimicrobials** that target disease causing bactria
* provide more efficiently produced **food**
87
Whats meant by **pharming?**
the process of genetically modifying plants and animals so they they produce substances whcih may be useful as pharmaceuticals
88
What animals can be used as bioreactors for pharming?
* chickens
* sheep
* goats
* pigs
* cows
89
The gene of interest is inserted into the animals using transgenic technology. Where is the transgene commonly expressed?
* eggs of chickens
* milk of cows, goats or sheep
90
Tell me what the promotor is of the following animals...
* sheep
* goat
* chicken
**Sheep:** Beta lactoglobulin milk promotor
**Goat:** Beta casein promotor
**Chicken:** Lysozyme promotor
91
What is **Human alpha antitrypsin (AAT)** used to treat?
How does it work?
* Used to treat cystic fibrosis
* is a plasma protein that inhibits elastase
* a key played in the inflammatory response that leads to tissue destruction
92
Whats the role of **ATII (anti thrombin III) ?**
Prevent blood clotting
93
What are the 2 types of gene therapy?
* Direct delivery
* Cell-based delivery
94
Tell me about **Direct delivery...**
* The therapeutic transgene is packaged into a delivery vehicle such as a virus
* It is then injected into patient
* It targets a specific organ e.g. liver
95
Tell me about **cell based delivery...**
* the therapeutic transgene is packaged into a delivery vehicle such as a virus
* the therapeutic transgene is introduced into a delivery cell such as a stem cell that is often derived from the patient
* the genetically modified cells e.g. stem cells are multilpied in a laboratory
* they are then readministered to the patient
* the target specific organs e.g. liver
