lecture 6 - applications of molecular techniques Flashcards
(14 cards)
What does RFLP-PCR stand for
Restriction Fragment Length Polymorphism PCR
what is it RFLP-PCR
A test where you cut DNA with enzymes to see if a mutation adds or removes a cut site—bands on a gel tell you the genotype.
What is ARMS-PCR
amplification-refractory mutation system
used for detection of mutations or SNPs
usually done by RFLP
Steps in a basic RFLP-PCR assay?
Design primers around the mutation
PCR amplify (~430 bp)
Digest with enzyme(s)
Run gel and compare band sizes
When to use ARMA-PCR instead of rflp-pcr
Use ARMS-PCR if no restriction site change exists; it uses allele‐specific primers to pick wild-type or mutant.
How does ARMS-PCR show WT vs. mutant?
WT reaction → 200 bp band
Mutant reaction → 150 bp band
Both have a 50 bp control band
What’s in an ARMS-PCR master mix?
PCR Buffer, Taq polymerase, dNTPs, control primers, WT-primer, mutant-primer, water, and DNA.
If both 200 bp+150 bp and 200 bp+50 bp show up, what’s the genotype?
You’re heterozygous—you have one WT and one mutant allele.
How do you detect a small deletion by PCR?
Design primers around deletion; WT gives a bigger band, deletion gives a smaller band, heterozygote shows both. Include a no-DNA control
What is multiplex PCR?
Running several primer pairs in one tube so you amplify multiple targets at once.
What are VNTRs?
Short DNA repeats (like 3 bp) that vary in number—useful for fingerprints and paternity tests.
How to calculate VNTR PCR product size?
Product = flanking length + (repeat length × copy number). E.g., 200 bp + (3 bp×20) = 260 bp.
How VNTRs help in paternity testing?
Child inherits one band from each parent—match bands to find the father.
How spot maternal contamination in CVS?
Fetal sample should show one maternal + one paternal allele. If you see two maternal alleles, it’s contaminated.
