DNA techniques
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chronological order of DNA technology development
• double helix model for DNA structure (based on x-ray results) • DNA polymerase • existence of DNA restriction nucleases, leading to their purification and use in DNA sequence characterisation. • DNA ligase • DNA cloning techniques • DNA-sequencing methods • polymerase chain reaction (PCR)
DNA ligase and how does it work it plays a role in ___
facilitates the joining of DNA strands together by catalysing the formation of a phosphodiester bond plays a role in repairing single-stranded breaks in duplex DNA, may repair double-strand breaks
restriction enzymes
used to cut DNA fragments of interest from one source to be ligated into a similarly cut plasmid vector
cloning vectors and plasmid vectors
cloning vectors - any DNA molecule used to carry a foreign DNA to a host cell plasmid vectors - a small, circular DNA molecule, derived from larger plasmids from bacterial cells
process of cloning with a plasmid vector
cut DNA and a plasmid with the same restriction enzymes (nucleases) and ligate them together with DNA ligase to form recombinant DNA
discovery of polymerase chain reaction was enabled by the use of ___, which is from a ___
the use of Taq polymerase, a DNA polymerase from a thermophilic bacterium
polymerase chain reaction (4 steps)
• mixture is heated to separate DNA strands • when the temperature is lowered, primers hybridise to DNA strands and synthesise complementary bases of the desired segment of DNA • process repeats in the next cycle, producing strands of gene of interest with different lengths • over time the specific sequence of interest accumulates
chain determining bases dictates
what base the sequence ends at during DNA sequencing
how different products are produced in DNA sequencing
different chain determining bases are used in separate reactions so that each produces a different set of products that terminate at different points in the sequence
how is DNA determined from electrophoresis in PAGE
reading off the bands in order
method used to separate products in DNA sequencing it analyses ___
electrophoresis in a polyacrylamide gel (PAGE) analyses the fluorescent labels
what is cDNA and how is it produced?
complementary DNA produced by reverse transcribing mRNA template using reverse transcriptase
cDNA synthesis from mRNA
• an oligo primer complementary to the 3’ end of mRNA is hybridised to the polyA tail of mRNA • reverse transcriptase is added to transcribe mRNA and produce a RNA/DNA hybrid • RNAse H introduces gaps in the RNA strand, forming fragments of RNA that serve as primers for DNA polymerase • DNA polymerase then copies the remaining single stranded cDNA into a double stranded cDNA
2 differences between DNA and cDNA
DNA contains segments of introns and exons cDNA only contains exons and has a polyA tail
reverse transcriptase is a ___ used in ___ where ___ is produced from ___
enzyme used in reverse transcription where a complementary DNA (cDNA) is produced from creating complementary bases of a mature mRNA
function of RNAase (ribonuclease)
breaks phosphodiester bonds of an RNA strand
PCR polymerisation chain reaction is used to ___
detect the presence of a viral genome in a nasal sample
illuminate sequencing is based on basic principles of ___
automated dideoxy sequencing
the process of illumina sequencing (3 steps)
• large sample of interest is broken into millions of short fragments • fragments are attached to a glass surface and amplified by PCR to generate DNA clusters (about 1000 copies of a single DNA fragment) • taking snapshots of the glass slide as reactions progress allow the determination of the sequence of nucleotides
BLAST searches in DNA/protein sequencing helps determine ___ and does what
sequence and finds similar/identical sequences in the database
purifying proteins by centrifugation separates ___ into ___ and ___
(once the plasmid vector has been inserted to the host and protein is generated, cells are lysed) used to isolate a particular part of the cell by separating cell extract into soluble and insoluble components (supernatant and pellet)
are proteins generally found in the supernatant or pellet after centrifugation
supernatant
4 methods of mechanically breaking open cells and tissues
• ultrasound forces cells through a small hole using high pressure • use mild detergent to make holes in the plasma membrane • cells are forced through a small hole using high pressure (French press) • shear cells between close-fitting rotating plunger and the thick walls of glass vessel
homogenisation is a procedure to
gentle mechanical procedure to break open cells and tissues