defining the genome II - DNA Flashcards
what is the chain-termination method?
method to determine the nucleic acid sequence of a purified DNA fragment (target)
what do you need for the chain-termination method?
target DNA
an oligonucleotide primer of approximately 20 nucleotide complementary to part of that DNA
DNA polymerase
mixture of deoxyribonucleotides (dNTPs) and dideoxyribonucleotide (ddNTPs)
what are restriction enzymes?
endonuclease enzymes that cut double stranded DNA at specific sequences that are called restriction site (4 to 8 pairs)
what do endonuclease do?
cleave phosphodiester bonds to leave free terminal 3’-OH and 5’-phosphate groups
what do exonuclease do?
cleave bonds at DNA ends
where do restriction enzymes come from?
come from bacteria
what is plasmid?
circle of DNA that replicates in bacteria independently of the bacteria chromosome and acts as a vector for delivering that DNA into target cells
what is southern blotting?
method to analyse DNA where mixtures of DNA fragments are separated by electrophoresis through an agarose gel and blotted onto a nylon membrane. Then a specific sequence can be detected using a DNA probe that is radioactively of fluorescently labelled
what is polymerase chain reaction (PCR)? why is it used for?
method to quickly generate microgram quantities of a particular DNA fragments from limited amounts of a complex DNA sample
establish the presence or the absence of a target sequence in a sample
what is next generation sequencing?
NGS can sequence millions of short DNA fragments simultaneously in a massively parallel fashion without the need of individual fragment isolation
what is NGS used for?
whole genome sequencing
targeted sequencing
transcriptome sequencing (RNAseq): sequencing of DNA reverse transcribed from RNA transcripts
CHIPseq: antibody to protein of interest is used to purify chromatin containing that protein prior to whole genome sequencing
why is most of the DNA in protein-coding genes, not coding?
because of its 5’ and 3’ untranslated regions (UTRs), enhancer and promoter sequences and its long introns
how can humans use genes in a more versatile way than other simpler species?
alternative splicing which allows a single gene to generate multiple mRNA isoforms and therefore multiple protein isoforms
size of the average human gene
27kb with 10.4 exons
average size of an exon
178 bp
