PCR Flashcards
Uses of PCR
DNA fingerprinting, classification of organisms, genotyping, clinical diagnostics, detections of pathogens, genetic cloning,
PCR primer sequences
should always be found on opposite strands, and oriented towards each other
DNA primers anneal to
complementary strands
variations in the size of an unstable allele in huntingtons disease.
causes variable expressivity.
PCR can be used to amplify short pieces of DNA
AND size differences can be visualised by gel electrophoresis.
DNA amplification by molecular cloning
steps are:
- DNA fragmentation, small enough to fit in vector
- Ligation in vector, plasmids or YAC
- Transformation: introduction of vector in bacteria or yeast
- culture and screen: antibiotic resistance
Recombinant plasmids are amplified in and purified
from a host cell
Recombinant plasmids are amplified in and purified
from a host cell
Cloning vector requirements
convenient restriction sites
Autonomous replication
selectable marker
Some plasmid vectors are manipulated to permit screening for recombinants (lacz for blue white screening)
Genomic libraries contain clones, which, together
represent the entire genome
Genomic DNA is cut with
a restriction enzyme
cDNA libraries represent
genes which are expressed
Colony hybridisation allows
purification of the clone of interest
The ideal genome editing tools would be
Edit any genome tools, high efficiency, high DNA sequence specificity, no undesired by- products
Cas9 is an
RNA guided DNA endonuclease: Cas9 endonuclease forms a complex with a guide RNA and localises to a target DNA sequence following a gRNA: genomic DNA pairing.
Restriction fragment length polymorphisms can
distinguish between 2 alleles (hetero and homo zygous)
Restriction fragment length polymorphisms are
abundant throughout eukaryotic genomes. RFLP’s result from differing positions of restriction sites
The bands on the gel when RNA is run represents
the small and big ribosomal RNA subunit
Probes from a variety of sources will
hybridise if the sequence similarity is high
- polymorphic alleles
- related genes or sequences from same species
- same sequence different species
Northern analysis reveals
gene expression data
RNA sequences can be used for
alternative gene spliced transcripts, post translational modifications, differences in gene expression in the whole transcriptome,
