Lecture 8 - Recombinant DNA Technology and DNA techniques

What is recombinant DNA technology?

combining dna from different sources into one molecule in vitro

Why is recombinant DNA technology used?

copy genes, study function and expression, produce useful proteins

What is a plasmid?

small circular dna molecule that replicates inside a bacterial host cell

What is the role of a cloning vector?

carries a gene of interest into a host cell for copying

What do restriction enzymes do?

cut dna at specific recognition sequences, producing sticky ends

What are sticky ends?

short single stranded dna ends that pair with complementary ends

What does DNA ligase do?

forms covalent bonds that joins dna fragments and seals the backbone

What are the main steps of gene cloning?

insert gene into a plasmid, put plasmid into bacteria, grow bacteria to copy the gene

What is the role of an antibiotic resistance gene in a plasmid?

helps identify bacteria that contain the plasmid

What does a promoter do?

help initiate transcription of a gene

What is PCR used for?

making millions of copies of a targeted dna region

What four components are needed for PCR?

template dna, heat stable dna polymerase, dntps, two primers

What happens during PCR denaturation?

heat separates double stranded dna into single strands

What happens during PCR annealing?

primers bind to complementary sequences on dna templates

What happens during PCR extension?

dna polymerase adds nucleotides to extend the primers

Why does PCR require heat-stable DNA polymerase?

to remain functional through high temperature cycles

What is gel electrophoresis?

method that separates dna fragments by movement through a gel in an electrical field

Which DNA fragments travel farther in a gel?

small fragments travel farther than large fragments

What is a DNA ladder used for?

estimating dna fragment sizes by comparison of fragments with known lengths

What does DNA sequencing determine?

order of nucleotides in a dna molecule

How does Sanger sequencing terminate DNA synthesis?

fluorescently labelled ddntp added, stopping strand extension

How are DNA fragments read in Sanger sequencing?

separate fragments by size and detect the fluorescent label on each fragment

What is massively parallel sequencing?

sequencing millions of dna fragments simultaneously

What is sequencing by synthesis?

identifying nucleotides as they are added to a growing strand

What are the four main steps of whole-genome sequencing?

extract and prepare dna, sequence dna, align the reads, identify genetic variants

What is bioinformatics?

using computational methods to analyse large biological datasets such as dna sequences