Recombinant DNA And Biotechonology 11-17 Flashcards
Primer design and construction
We need to know the sequence of two small flanking regions of the sequence we wish to amplify
Denaturing the target protein in PCR
Produce single stranded DNA (will allow the primers to bind when cooled
-heat is used
What is the primer for PCR?
DNA
Annealing primers in PCR
DNA sample is cooled and the primers bind to the complementary sequence in the target DNA
Chain extension in PCR
A DNA polymerase uses dNTPs to extend the primer and build a complementary copy of the DNA strand
Steps _______ repeated 20-30 times (denature, anneal, extend)
2-4
Why do you need to use heat stable Taq DNA polymerases?
So its stable at higher temps
Advantages of PCR
- sensitivity
- speed
- DNA can be used for gel electrophoresis, southern blotting, etc
- mutation detection, detection of latent viruses, forensics, prenatal genetic diagnosis
Northern blot
- mRNA
- quantitative AND qualitative
- expression of gene
- only one gene at a time
Micro arrays
- mRNA levels
- contain thousands of immobilized sequences (probes) on glass slides
- compare global gene expression changes in different cell types
- synthesize and label cDNA from two different cell types with different fluorescent probes
- computer analyzed
Proteins-proteomics
Similar to genomics but looks at all the different proteins produced in particular cell or tissue types (including post-translational modifications, enzyme modulation by phosphorylation , etc..
-more for research, not clinic
ELISA (enzyme linked immunosorbent assays)
- antigen bound to the well of a microtiter
- probed with an antibody linked to an enzyme
- detect by adding substrate for enzyme to form a colored reaction
False positive for ELISA
Sometimes you pick up a false positive
- if negative, you can feel confident about results
- if positive, follow up to make sure its not false positive
Western blot
- proteins
- samples separated by gel electrophoresis based on size
- proteins blotted onto membrane
- probed with an enzyme-linked antibody to identify bands
- more specific than ELISA, but more time and labor intensive, less sensitive
Analysis of DNA
- southern blot
- ASO
- PCR
analysis of RNA
- northern blot
- microarray
- PCR
Analysis of protein
- western blot
- ELISA
- proteomics
Purpose of southern blot
Detects DNA changes
Purpose of northern blot
Measures mRNA amounts
Purpose of western blot
Measures protein amounts
Purpose of ASO
Detects DNA mutations
Purpose of microarray
Measures many mRNA levels at once
Purpose of ELISA
Detects proteins (antigens) or antibodies
Purpose of proteomics
Measures abundance, distribution, posttranslational modifications, functions, and interactions of cellular proteins
Quantitative techniques
- northern blot
- western blot
- microarray
- ELISA
- proteomics
- sometimes PCR
Is DNA quantitative?
No
Sickle cell mutation
B-globin gene that eliminates restriction site for restriction enzyme
What does sickle cell create?
An RFLP: in this case, the disease causing mutation creates an RFLP that can be used for diagnosis (one larger fragment of DNA)
In most diseases, an RLFP used for diagnosis ones what?
Is linked to the disease, but no the actual disease causing mutation
RFLP in sickle cell
Enough DNA moving around you usually carry passenger polymorphism during crossover, not the case for sickle cell
Restriction enzyme for sickle cell
Abolished
Sickle cell and southern blot
With probe to beat-globin gene
Normal cells vs sickle cells on southern blot
- normal is smaller band
- sickle cell is larger banded
Genetic testing for sickle cell: alternative
Could do a similar procedure using PCR, amplify the region containing the potential mutation, digest PCR products with MstII
PCR for sickle cell
- use primers flanking beta globin gene, or region where mutation may be
- digest PCR product with MSTII, run on gel
- larger band for sickle cell
ASO and sickle cells
ASO probes-Allen specific oligonucleotides
VERY SPECIFIC
-very reliable and specific, very fast, minimal complications with the technique, non-invasive, inexpensive
