lecture 4 types of PCR Flashcards
what you would put in PCR tube
DNA template
Primers (forward and reverse)
nucleotides
taq polymerase
buffer (tris HCL pH 8.0)
MgCl2
PCR
denature (95 degrees)
anneal (50-60)
extension (75)
uses of PCR
genotyping the patient
copying the pathogen
phenotyping the disease
genotypic the patient
diagnosis of genetic traits
detection of carriers of genetic traits
tissue matching (HLA typing - human leukocyte antigen, proteins on the outside of cells shows that cells arent foreign)
predicting reponse to drugs
genotypic the pathogen
diagnosis of species and strain of infecting pathogen
phenotyping the disease
measuring disease progression
measuring disease severity
sources of DNA for PCR
blood, hair, buccal smear (more cheek) , cells from amniotic fluid (fluid surrounding the featus)
2 PCR techniques for genotyping the patient
PCR-RFLP (restriction fragment polymorphism)
ARMS-PCR (amplification refractory mutation system)
PCR-RFLP
- amplify the substrate
- add the RE that cuts the sequence in the allele you’re interested in (allele 2)
if the the RE site is in neither allele - homozygous allele 1
if RE site in both alleles - homozygoes allele 2
if RE site in one of the allele - heterozygous
disadvantages of PCR-FRLP
only possible if the site contains a known RE site
some RE are expensive
ARMS-PCR
detects alleilic variants using allele specific primers
presence or absense of a PCR product is diagostic for the presence or absence of the target allele
clinical example of PCR-FRLP
diagnosis of sorsbys fundus dystrophy
clinal example of ARMS-PCR
diagnosis of cystic fibrosis
RFLP vs ARMS
RFLP:
uses locus specific primers
relies of the presence or absence of a restriction site to distinguish between variants
ARMS:
uses allele specific primers
relies of the stringency of the PCR to distinguish between alleles
genotypic the pathogen
DNA/RNA can be obtained from
blood, sputum, urine, faeces, skin swab, tissue biopsy
