PCR and microbiomes Flashcards
steps of the PCR thermocycler program
- Denaturation
- Annealing
- Elongation
how to calculate the number of copies of a given amplification
grows exponentally
essential components in the reaction mixture
- primer-(FV)(FC)=(SC)(X)
- buffer- (FC)(FV)=(SC)(X)
- NTPs- same as 5
- polymerase-same as 5
- template DNA-fix measurements and repeat 1
- water- added volumes-FV
main considerations for designing PCR primers
denaturing heat and where it cuts the gene so not to lose some of it.
how to use PCR to introduce point mutations and restriction sites into a gene
primers designed to be come restriction sites or promoters
the applications of PCR
forensics evaluate cancers ID specific alleles Detection of infectious agents studying evolution
determination of the Random Match probability.
multiply the probabilities together
VNTR
variable number tandem repeat (or VNTR) is a location in a genome where a short nucleotide sequence is organized as a tandem repeat.
STR
Short Tandem Repeats (STRs): regions in the genome where short stretches of sequences are repeated. The number of repeats can vary per individual.
RFLPs
Restriction Fragment Length Polymorphisms (RFLPs) – amplify regions of DNA with a known restriction site
troubleshoot issues with PCR reactions
possibility of errors and those errors repeat in those copies
the basic set up of a Chip
sticky pieces that the DNA can attach to and will light up in the color given to it, there a some spots set to always light up
differences between DNA and RNA-based arrays
Identifying which genes are being expressed (RNA). DNA looks at the whole genome.
Comparative Genomics
a field of biological research in which the genomic features of different organisms are compared.
Data Mining
“the nontrivial extraction of implicit, previously unknown, and potentially useful information from data”
Phenomic
The Phenotypes that result from the genome and the proteome
Proteomics
Identification of all the proteins expressed in every cell of an organism
Transcriptomics
the study of the transcriptome—the complete set of RNA transcripts that are produced by the genome
Bioinformatics
the use of computer programs to find things like homologs and primers
Mapping
Mapping revealed that genes are not evenly dispersed through the genome, but are clustered
Annotation
the process of identifying regions in the sequence
Molecular Clocks
a technique that uses the mutation rate of biomolecules to deduce the time in prehistory when two or more life forms diverged
Housekeeping genes
typically constitutive genes that are required for the maintenance of basic cellular function, and are expressed in all cells
Neutral regions
DNA segments without active roles in the cell will be a reflection of the frequency of random mutation
Northern blots
mRNA is isolated from a tissue and run on an Agarose gel
The mRNA fragments are transferred from the gel to a 2-D membrane.
The membrane is incubated with a labeled DNA probe that will hybridize to the mRNA that contains its complementary sequence.
Microbiome
comprises all of the genetic material with a Microbiotia
Microbiotia
the entire collection of microorganisms in a specific niche
Amplicon sequencing
amplify a highly conserved gene (eg 16S rRNA) to get sequence for all of the different bacteria in the microbiota.
Metagenomic sequencing
no amplification, just isolate DNA and start sequencing
denaturing
the Double-stranded DNA template must be melted apart to 2 single strands.
annealing
the Primers have to base-pair with the complementary DNA on the template
elongation
Initiation – The initial reaction is heated for several minutes to destroy any other enzymes in the reaction mix.
Final elongation – after the last cycle, the reaction is held at the elongation temperature to let any incomplete fragments finish
southern blot
Use nucleases to cut up the genomic DNA into smaller fragments.
Separate the fragments by size on an agarose gel
Transfer the DNA fragments from the gel to a 2-D membrane.
Incubate with a labeled DNA probe that will hybridize to the fragment of DNA that contains its complementary sequence.
