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Bio 114 Lecture 5
cDNA Cloning and Recombinant Proteins
43
Biology
Undergraduate 1
02/28/2013

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Term

True or false?

cDNA is a manmade product?

Definition

True

 

it is complementary DNA that is a copy of mRNA. the enzyme Reverse transcriptase is needed to make cDNA

Term

True or False?

mRNA can be cloned directly?

Definition

False

 

it cannot be cloned directly, only double stranded DNA can be clone thus the need to make cDNA from mRNA.

Term

why do we want cDNAs? (2)

 

Definition
  • Enrichment - cDNA only represents expressed part of the genome (no introns)
  • cDNA can be used to express recombinant proteins in heterologous systems (as it is uninterrupted by introns)
Term
name two ways we find the clone containing our sequence of interest
Definition

DNA Hybridisation

 

Based on the properties of the encoded protein such as immunodetection and complementation cloning

 

the choice of method depends on what we know about the target

Term
what is needed to detect clones by hybridisation?
Definition

a hybridisation probe - this is a DNA/RNA fragment (100-1000 bp)

  • specifically hybrised to traget DNA/RNA sequence
  • labelleled/tagged so its location can be visualised
Term
what sort of DNA can be used in probe making?
Definition

cDNA specifically the corresponding sequence to our sequence of interest

 

Term
what is a degenerate oligonucleotide?
Definition

it is a mixture of different sequences.

 

if the protein sequence is known then we can deduce all the possible nucleotide sequences it could encode and select the least degenerate 20 base region.

Term
in radioactive probe labelling phosphorus is used. 32P and 33P. What are the advantages and disadvantages of this method?
Definition

advantages - radioactive probes can be detected directly by autoradiography

 

disadvantages - saftey, complicated handling procedure, short half-life and limited detection methods

Term
what are the half lives for 32P 33P?
Definition

32P 14.3 days

 33P 25.4 days

Term
what is used in non-radioactive labelling of probes?
Definition

Digoxigenin (DIG)

 

this is a highly immunogenic steroid and can be conjugated to nucleotides

Term
what are the advantages and disadvantages of using digoxigenin to label probes?
Definition

Ads - digoxigenin probes are safe and stable#

Disadvantages - detection methods are more complex and more complex involving more steps

Term
detection of DIG label is a 2 step process. what are they?
Definition

DIG label recognised by specific antibodies bound to alkaline phosphotase enzyme

 

filter incubated with AMPPD, a dioxetane that is cleaved by alkaline phosphotase in a reaction that produces chemiluminescence. this is captured on film only where the antibody is bound

Term
what is unusual about the enzyme terminal transferase that is used in the labelling of gene probes?
Definition

it does not require a DNA template

it is used used to add polynucleotide tails to the 3' end of DNA molecules and works best when the 3' end is not recessed (ie, blunt-end or 3' overhang)

Term
taq polymerase and the PCR reaction can be used to label gene probes but what must the reaction be carried out in the presence of?
Definition
in presence of a labelled dNTP (e.g. dTTP coupled to DIG)
Term
when using immunodetection to identify clones what must we do to the protein first?
Definition

we must purify the protein of interest.

 

then we can raise specific antibodies against the purified protein

Term
when using antibodies for clone identification, how many types of antibodies are used?
Definition

2 - primary and secondary (radiolabelled) antibody

 

  • when proteins are bound to nitrocelluose filter, they are incubated with the primary antibody then the filter is washed.
  • then the filter is uncubated with the secondary radiolabelled antibody. this identifies specifi plaques to then which autoradiography is performed
Term
describe the steps in using a histidine auxotroph of E.coli to clone a cDNA encodig an enzyme required for histidine
Definition

1 ligate plasmid and cDNA frgaments

2. Transform into the His- bacteria

3. transfer to medium lacking histidine

4. colonies on the plate grow as transformed plasmid has provided gene for biosynthesis of histidine

Term
why do we use recombinant DNA technology?
Definition

because isolation of sufficient amounts of most proteins is difficult because many proteins are expressed at low levels

the ability of the source tissue may be very limited (e.g. for proteins for therapeutic use such as insulin)

Term
when expressing foreign proteins in E.coli what must the DNA be that is inserted into the bacteria?
Definition

cDNA (no introns)

the inserted cDNA must also be adjacent to appropiate sequences for its transcription and translation in the host cell i.e.

promoter

ribosome binding sequence

transcription terminator

Term
obtaining a high level of expression in E.coli is dependant on which factors? (4)
Definition

1. using a high copy number plasmid vector

2. using an inducible promoter

3. efficiency of translation in the host organism

4. ensured stability of the foreign protein product in the host organism (eg. by making fusion proteins)

Term
why can we not use a constituently expressed promoter?
Definition
because recombinant protein is often toxic for bacteria so there is slow growth and strong selection pressure for elimination or rearrangement of the plasmid
Term
how is the lac promoter used to control expression of foreign proteins?
Definition
  • IPTG is a stable analogue of lactose. It binds to the lac repressor protein
  • Lac repressor IPTG complex no longer binds lac operator
  • Addition of IPTG to the culture induces expression from the lac promoter
Term
what does IPTG stand for and what is it?
Definition

Isopropyl β-D-1-thiogalactopyranoside

 

This compound is used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon. Unlike allolactose, the sulfur (S) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from metabolizing or degrading the inductant; therefore the IPTG concentration remains constant

Term
in codon optimisation strategies how can be overexpress low abundance tRNAs?
Definition
use an engineered host cell
Term
how can we alter the sequence of the gene of interest to match donor codons to the codons most frequently used in the host organism?
Definition
by chemically synthesising a new gene
Term

recombinant DNA technology:

what is the use of Brain-deprived neurotropic factor?

Definition
stimulates regrowth of brain tissue in patients with Lou Gehrigs' Disease
Term

recombinant DNA technology:

what is the use of Colony-stimulating factor?

Definition
stimulates production of white blood cells in patients with cancer and AIDs
Term

recombinant DNA technology:

what is the use of Erythpoietin?

Definition
prevents anemia in patients undergoing kidney dialysis
Term

recombinant DNA technology:

what is the use of Factor VIII?

Definition
replaces the clotting factor missing in patients with hemophilia A
Term

recombinant DNA technology:

what is the use of Growth hormone?

Definition
replaces missing hormone in people with short stature
Term

recombinant DNA technology:

use of insulin?

Definition
stimulates glucose uptake from blood in some people with diabetes
Term

recombinant DNA technology:

use of platelet-derived growth factor

Definition
stimulates wound healing
Term

recombinant DNA technology:

use of tissue plasminogen activator?

Definition
dissolves blood clots after heart attacks and strokes
Term

true or false

vaccine proteins such as Hep B, herpes, influenza, lyme disease, meningistus, pertussis are all made from

recombinant DNA technology?

Definition

true

they prevent and treat infectious diseases

Term
name two types of DNA used as probes
Definition
cDNA and degenerate oligonucleotides
Term
name the method used to detect the location of a radioactively labelled probe on a filter?
Definition
autoradiography
Term
name a reagent used in non-radioactive labelling?
Definition
DIG - digoxygenin
Term
name the enzyme conjugated to the antibody used in the non-radioactive detection method
Definition
alkaline phosphatase
Term
name the process that produces light in the non-radioactive detection method
Definition
chemiliminescence
Term
name 2 enzymes used to label DNA
Definition

terminal transferase

Taq polymerase

Term
name the synthetic comound that binds to the lac repressor
Definition
IPTG
Term

name two usedful features of expression vectors for high level expression

 

Definition

high copy number

inducible promoter

Term
name 3 elements needed adjacent to a cDNA for expression in a bacterial plasmid
Definition

promoter

ribosome binding sequence

transcription terminator

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