Molecular Biology Techniques

what is recombinant dna technology?

-allows manipulation of dna sections such as genes by isolating them and creating multiple identical copies (clones)

what are restriction enzymes?

-molecular scissors that recognise short specific nucleotide sequences (4, 6 or 8 bp)

-cut both strands of the dna sugar-phosphate backbone

where were restriction enzymes first found and what do they do naturally?

-found in bacteria where they protect against viral infection by cutting viral dna

what are cohesive (sticky) ends?

-single-stranded overhangs left after a staggered restriction enzyme cut

-allow complementary fragments to anneal via hydrogen bonds

what enzyme permanently joins two annealed dna fragments?

-dna ligase, which forms a covalent bond between the free -oh and -po4 groups

what is a cloning vector?

-a dna molecule (e.g. a plasmid) that carries a foreign dna fragment into a host cell and self-replicates

what are the three key features of a plasmid cloning vector?

-origin of replication (ori)

-antibiotic resistance gene

-multiple cloning site (mcs)

what is the purpose of the antibiotic resistance gene in a plasmid?

-allows only bacteria containing the plasmid to survive in the presence of that antibiotic (selective growth)

what is the multiple cloning site (mcs)?

-region on a plasmid with clustered restriction enzyme recognition sites where dna fragments can be inserted

how is a recombinant plasmid created?

-dna fragment and plasmid are cut with the same restriction enzyme

-mixed so cohesive ends anneal,

-then sealed by dna ligase

what is transformation?

-process by which bacteria (e.g. e. coli) take up a recombinant plasmid

how many plasmid copies are generated per bacterium?

-approx 100–200 copies using the plasmid's ori

what is blue-white selection?

-method to identify recombinant bacteria

-insertion of dna into the lacz gene disrupts it

-colonies appear white instead of blue

what is a genomic library?

-collection of overlapping cloned dna fragments representing an entire genome

what is an expression vector?

-plasmid vector containing regulatory elements (e.g. promoters, translation initiation sites)

-needed to produce protein from a cloned gene

why is cdna used to express eukaryotic proteins in bacteria?

-eukaryotic genes contain introns which bacteria cannot proces

-cdna is made from mrna so lacks introns so bacteria can express it

what is cdna and how is it made?

-complementary dna

-dna copy of an mrna molecule produced using the enzyme reverse transcriptase

what is a cdna library?

-collection of plasmids containing cdna

-inserts representing the mrnas expressed in a particular tissue at a specific time

how does a cdna library differ from a genomic library?

-cdna library contains only expressed genes and lacks introns

-genomic library represents the entire genome including non-coding regions

what is nucleic acid hybridisation?

-method to identify specific nucleic acid sequences

-by exploiting base-pairing complementarity between single-stranded nucleic acids

what is a dna probe?

-labelled single-stranded dna molecule used to detect specific complementary dna or rna sequences in a mixture

how can dna probes be labelled?

-using radioactive isotopes (32p)

-fluorescent molecules

-enzyme-conjugated labels

=affinity labels such as biotin or digoxigenin

what is southern blotting?

-technique that identifies which restriction fragment of genomic dna contains a sequence of interest

-using a labelled dna probe

what are the key steps of southern blotting?

-digest dna with a restriction enzyme

-separate by gel electrophoresis

-denature to single strands

-transfer to membrane

-hybridise with probe

-detect label

what is northern blotting?

-similar to southern blotting but detects rna

-most often used to determine whether a gene is expressed in a particular tissue

what is in situ hybridisation (ish)?

-detects specific rna molecules directly in cells or tissues

-using probes that hybridise to mrna at its site of expression

what is fish?

-fluorescent in situ hybridisation

-uses fluorescently labelled probes to locate gene expression in tissues or genes/sequences on chromosomes

what is sanger sequencing?

-dna sequencing method developed by fred sanger (nobel prize 1980)

-uses ddntps to terminate strand synthesis and generate fragments of all possible lengths

what are ddntps and why do they terminate dna synthesis?

-dideoxynucleotides that lack a 3'-oh group

-without this dna polymerase cannot form the next phosphodiester bond, stopping extension

how is the sequence read in automated sanger sequencing?

-fragments are separated by capillary electrophoresis

-each ddntp has a different fluorescent colour

-detected by a laser as fragments pass through

what is next generation sequencing (ngs)?

-massively parallel sequencing of millions of short dna fragments simultaneously

-producing gigabases of data per run at low cost per base

how has the cost of sequencing the human genome changed?

-original human genome project cost ~$2 billion over 10 years

-it can now be done for under $1,000

what are snps?

-single nucleotide polymorphisms

-single base pair differences between individuals that can affect disease risk and drug response

what is pcr?

-polymerase chain reaction

-technique to selectively amplify a specific dna sequence using repeated cycles of heating and cooling

what is taq polymerase and why is it used in pcr?

-thermostable dna polymerase from thermus aquaticus

-remains active at the high temperatures used in pcr

what are primers in pcr?

-short single-stranded dna sequences

-flank the target region and are extended by taq polymerase

what are the three steps of a pcr cycle and their temperatures?

-denaturation (95°c) — separates dna into single strands

-annealing (45–68°c) — primers bind to template

-extension (72°c) — taq polymerase synthesises new dna

how many copies result after 30 pcr cycles?

-approximately 1 billion copies (2³⁰ ≈ 10⁹)

what are the main limitations of pcr?

-requires prior sequence knowledge for primer design

-highly sensitive to contamination

-best suited to short-to-medium fragments (~10 bp to ~50 kbp)

what are the main applications of pcr?

-detecting mutations and pathogens in diagnostics

-forensic identification

-amplifying dna from trace samples

-food species testing

what are strs and how are they used in pcr-based genotyping?

-short tandem repeats

-repetitive sequences that vary in length between individuals

-pcr amplifies the str region

-gel electrophoresis separates alleles by size for identification or clinical prediction