analytical methods in molecular genetics Flashcards
ankush prashar (10 cards)
what is genome sequencing?
determining the base sequences of an entire genome
what is genome sequences used for?
- comparing species and tracing evolutionary relationships
- comparing individuals to identify mutations
- identifying genes for particular traits such as genes associated w diseases
what is the Human Genome Project?
a project proposed in 1986 to determine the normal sequence of all human DNA
what is the Sanger n Coulson method?
a termination method, which uses dideoxynucleotides/ddNTPs (ddATP, ddCTP, ddGTP and ddTTP) for early terminationof DNA polymerisation
what is required for the Sanger n Coulson method?
- single stranded DNA that is to be sequenced
- primer
- dNTPs
- labelled ddNTPs
- DNA polymerase 1
what is high throughput/next generation sequencing?
- parallel sequencing of millions of short DNA fragments in liquid or solid matrix
- cost efficient
- allows for sequencing of large genomes n transcriptiomes
what is illumina NGS?
illumina next generation sequencing:
- sequencing by synthesis using DNA polymerase and fluorescent nucleotides
explain the PCR reaction of illumina NGS.
- DNA cut into fragments mechanically or w enzymes
- fragments denatured using heat
-> separates strands - short, synthetic oligonucleotides attached to each end, these attached to a solid support
- fragments amplified by PCR
explain how the DNA is sequenced following the PCR of an illumina NGS
- universal primers, DNA polymerase and dNTPs tagged w fluorescent dyes are added
- one nucleotide added to the new DNA strand in each cycle, unincorporated dNTPs removed
- fluorescent colour of new nucleotide at each location detected by camera
- fluorescent tag removed from nucleotide, labelled synthesis cycle repeats
what is a transcriptome?
subset of the genome expressed as RNA in a particular cell or tissue at a particular time
