PCR - Clinical Uses
- Prenatal diagnosis
- Detection of mutated oncogenes
- Diagnosis of infections
- Forensics
Adv = sensitivity! - only one strand of DNA is needed to detect a particular DNA sequence
Reverse Transcriptase PCR
- Used to amplify DNA
- RNA is converted to DNA by reverse transcriptase
- Gene expression in the form of mRNA (rather than DNA sequence) can therefore be analysed
PCR Process
Polymerase chain reaction is a molecular genetic investigation technique
Prep:
- requires 2 DNA oligonucleotide primers, which are complimentary to specific DNA sequences at either end of target DNA
- DNA sample added to test tube along with the 2 DNA primers
- a thermostable DNA polymerase (Taq) is added
PCR Cycle:
- mixture is heated to almost boiling point causing denaturing (uncoiling) of DNA
- mixture is then allowed to cool - complimentary strands of DNA pair up as there is an excess of primer sequences they pair with DNA preferentially
> Cycle repeated w amount of DNA doubling each time !
Molecular Biology Techniques
SOUTHERN BLOTTING = Detects DNA
NORTHERN BLOTTING = Detects RNA
WESTERN BLOTTING = Detects proteins - uses gel electrophoresis to separate native proteins by 3D structure
SNOW (South > North > West)
DROP (DNA > RNA > Protein)
ELISA = Enzyme-linked immunosorbent assay
- a type of biochemical assay used to detect antigens and antibodies
- a colour changing enzyme is attached to antibody if looking for antigen and to an antigen if looking for an antibody
- sample therefore changes colour if either is detected
