True or false?

cDNA is a manmade product?

True

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

True or False?

mRNA can be cloned directly?

False

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

why do we want cDNAs? (2)

• 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)

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

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

cDNA specifically the corresponding sequence to our sequence of interest

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.

advantages - radioactive probes can be detected directly by autoradiography

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

³²P 14.3 days

 ³³P 25.4 days

Digoxigenin (DIG)

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

Ads - digoxigenin probes are safe and stable#

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

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

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)

we must purify the protein of interest.

then we can raise specific antibodies against the purified protein

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

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

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)

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

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)

• 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

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

recombinant DNA technology:

what is the use of Brain-deprived neurotropic factor?

recombinant DNA technology:

what is the use of Colony-stimulating factor?

recombinant DNA technology:

what is the use of Erythpoietin?

recombinant DNA technology:

what is the use of Factor VIII?

recombinant DNA technology:

what is the use of Growth hormone?

recombinant DNA technology:

use of insulin?

recombinant DNA technology:

use of platelet-derived growth factor

recombinant DNA technology:

use of tissue plasminogen activator?

true or false

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

recombinant DNA technology?

true

they prevent and treat infectious diseases

terminal transferase

Taq polymerase

name two usedful features of expression vectors for high level expression

high copy number

inducible promoter

promoter

ribosome binding sequence

transcription terminator