38. the impact of science on medicine - recombinant DNA technology

Question 1

which approach is used to AMPLIFY a specific stretch of DNA

Answer 1

POLYMERASE CHAIN REACTION

(PCR) is a process where you DESIGN PRIMERS to BIND to SPECIFIC REGIONS of DNA then use Taq POLYMERASE to AMPLIFY the REGIONS in BETWEEN.
You can make thousands of COPIES of that one part of the DNA.
Combining it with REVERSE TRANSCRIPTASE (RT-PCR) allows you to AMPLIFY RNA.

Question 2

What are the (potential) advantages of SANGER SEQUENCE compared with NeXT generation sequencing?

Answer 2

• Cost effective for low target number
• Simple data analysis
• Longer reads

Sanger sequencing is currently the “gold standard” for single target validation.

The big advantage of NGS is that you have really high sequencing depth and much much higher discovery power. Indeed with Sanger you can only find what you look for, whereas NGS allows you to find everything. However, the analysis is much more challenging.

Question 3

what is RECOMBINANT DNA (how is it formed)

Answer 3

GENE OF INTEREST (cloned by PCR) is LIGATED (joined) with a VECTOR (plasmid)

enzymes used to cut ends
ligase enzyme used to join

• RECOMBINANT DNA - 2 different parts joined

Question 4

what happens with the RECOMBINANT DNA

Answer 4

introduce this plasmid into a cell
- LIPOSOMAL COATING to allow delivery (cross plasma membrane)

cell uses sequence in plasmid, promotor region, for TRANSCRIPTION to mRNA for TRANSLATION

to form RECOMBINANT PROTEIN
- can be PURIFIED

Question 5

how do you form MONOCLONAL ANTIBODIES

Answer 5

• INJECT mouse with dead virus or Recombinant Protein to STIMULATE ANTIBODY PRODUCTION
• ANTIBODY-FORMING CELLS in the SPLEEN are ISOLATED
• FUSED with TUMOUR CELLS (grown in tissue culture) to form HYBRIDOMAS
• ANTIBODY-PRODUCING HYBRIDOMAS are CLONED and SCREENED

Question 6

If you inject a human with mouse-derived monoclonal antibodies, what would
happen?

Answer 6

RAISE an IMMUNE RESPONSE
as FOREIGN

Question 7

why might mouse-derived Monoclonal Antibodies still be used clinically despite problems

Answer 7

• for SCREENING or DIAGNOSIS
• for use in an IMMUNOCOMPROMISED individual

Question 8

most clinically useful mABs are now..

Answer 8

Humanized

• VARIABLE REGION stays MOUSE-DERIVED
• REST if HUMANIZED

Question 9

most clinically useful mABs are now..

Answer 9

Humanized

• VARIABLE REGION stays MOUSE-DERIVED
• REST is HUMANIZED

Question 10

most clinically useful mABs are now..

Answer 10

Humanized

• VARIABLE REGION stays MOUSE-DERIVED
• REST if HUMANIZE

Question 11

how are mABs HUMANIZED (3)

Answer 11

• use HUMAN B CELLS (inject in volunteers instead of mice but ethical issues)
• use TRANSGENIC MICE expressing HUMAN IgG GENES (Xeromouse) (protein coding for human immunoglobulins inserted)
• in vitro PHAGE / YEAST DISPLAY methods (introduce DNA that encodes for these antibodies into OTHER CELL TYPES)

Question 12

MURINE antibodies - 0% human ending

Answer 12

-omab

Question 13

CHIMERIC antibodies - 65% human ending

Answer 13

• ximab

Question 14

HUMANIZED antibodies - 90% human ending

Answer 14

-zumab

Question 15

FULLY HUMANIZED antibodies - 100% human ending

Answer 15

-umab

Question 16

microRNAs bind the the 3’ end of an mRNA (mostly). What effect do they usually they have?

(MicroRNAs (miRNAs) are a class of non-coding RNAs that play important roles in regulating gene expression)

Answer 16

• suppress translation
• decrease mRNA stability

therefore produce LESS PROTEIN

Question 17

New Technologies

Answer 17

*Genetic analysis
- Genomic DNA sequencing… 5 million genomes

*Gene-based interventions
- miRNAs
(naturally ocurring - no immune response, specific, very small)