Chapter 12 Flashcards
Genetic engineering
technology that allows the genetic material of an organism to be manipulated through using modern molecular biology techniques - e.g. introducing, eliminating or changing DNA.
Genetic engineering is used because of…
economical reasons
Major steps of genetic engineering
isolation of genes of interest, insertion into transfer vector, transfer vector to the organism to be modified, transformation of the cells of the organism, selection of GMO
Genetically modified organism
an organism whose genetic make-up has been altered by artificial means
Restriction enzymes
One class of enzymes that can cut DNA into a reproducible number of fragments and which can occur naturally in micro-organisms, such as bacteria
Why are they called restriction enzymes
restricted to where they can cut
How are restriction enzymes labelled
letters = organism it came from, roman numerals = order extracted
Where do restriction enzymes originate from
e.g. immune system of bacteria
How do restriction enzymes work
- cuts made at specific points on a DNA strand
- either makes a blunt end or sticky end cut
Sticky ends are…
complementary
How do restriction enzymes cut
break phosphodiester linkages
Phosphodiester linkages
join between sugar and phosphate backbone
Each restriction enzyme has a specific…
recognition sequence
Recognition sequence
sequence of three to six nucleotides within a DNA molecule that forms the specific site for a restriction enzyme that can cut the DNA at that point; also termed a cutting site
Gel electrophoresis
technique for sorting through an electric field a mixture of DNA fragments (and other molecules with a net charge) on the basis of different fragment lengths
Gel electrophoresis purpose
to sort out DNA according to size
How does gel electrophoresis work
- DNA is placed at one end of a gel-like substance
- Blue tracking dye is added
- Gel is exposed to an electric field (i.e. an electric current is passed through it): negative end is where you place the DNA and positive end is the opposite pole
- DNA moves towards positive end (net negative charge)
- Fragments move through reptation
- Shorter fragments move more quickly and further than longer fragments
- Fragments are dyed with ethidium bromide (UV) so can see
Why do shorter DNA fragments travel further
fit through smaller pores, less resistance
Name of gel
agarose gel
What is gel placed in (blue solution)
methylene blue solution
How can one particular DNA fragment can be picked out from millions
probe
Probe
single-stranded segment of DNA (or RNA) with a base sequence complementary to that in a target strand of DNA and that carries a radioactive or fluorescent label to detect it.
How does a probe work
- it has a radioactive or fluorescent marker
- located particular sequence
- has a sequence complementary to that of the target DNA
Why does it have to be denatured
to become single stranded so it can be located
Ligase
enzyme that catalyses the joining of two double-stranded DNA fragments. Makes sugar-phosphate back bone bond permanent.
Ligase can form… (shape of DNA)
linear or circular DNA
Plasmids act… from central chromosome
act separately
Plasmids can…
cross membranes
Vectors
in genetics, refers to an agent such as a plasmid or a virus that carries passenger DNA into a cell.
Steps of vectors/recombinant DNA
- DNA of plasmid is cut using restriction enzyme (sticky ends)
- passenger DNA cut with same restriction enzyme
- passenger DNA is mixed with plasmid DNA
- ligase makes join permanent
- plasmids with passenger DNA are selected + placed in plasmid
Gene cloning
process of making multiple identical copies of a specific gene or segment of DNA
Gene cloning process
Gene to be cloned is inserted into a bacterial plasmid, the plasmid is then reinserted into the bacterial cell, plasmid may multiply up to 20 times within the cell= 20 copies of the gene, bacterial cell multiplies every 20 mins, within these new cells plasmids multiply again, cell divides etc.
What does PCR stand for
Polymerase Chain Reaction
PCR
technique for amplifying tiny amounts of DNA exponentially through many cycles of multiplication; sometimes termed ‘DNA xeroxing’
PCR purpose
to amplify DNA
PCR steps
- Denature
- Bind primers
- Extend primers
Denature (PCR)
DNA is heated to 94ºC for 2 minutes to separate into single stranded DNA
Bind primers
DNA heated to 55º for 2 minutes to add short segments of single stranded DNA that bind to either end
Extend primers
polymerase uses primers as a starting point to extend to form 2 complete strands. Must supply nucleotides. 72C for 1 min
Time length of each cycle (PCR)
5 minutes
Taq polymerase
used to synthesise DNA (PCR)
How is DNA extracted
amniocentesis, chorionic villus, blood cells/cheek cells
Chorionic Villus Sampling CVS
placenta section removed (pregnancy)
Conjugation
bacteria can transfer plasmids to other bacteria
Other ways of obtaining specific segments of DNA
synthesise DNA from nucleotide building blocks (DNA synthesiser), and make a copy of the DNA using an mRNA template (reverse transcriptase)
Reverse transcriptase
enzyme that directs the formation of copy DNA from a messenger RNA template
Transgenic organisms
organisms that carry in their genomes one or more genes artificially introduced from another species
DNA profiling
technique for identifying DNA from different individuals based on variable regions
Variable regions - STR, (aka micro-satellites)
Detection of variation uses…
a combination of single-locus probes, each specific to a different STR (locus LSTR)
STRs and HVRs
very variable between unrelated people
STR
chromosomal sites where many copies of a short DNA sequence are joined end to end
STR sequences are normally… bases long
2-4 bases long and the number of repeats are variable between unrelated people.
HVR stands for
hyper-variable regions
HVR
regions of chromosomal DNA in which great variation exists in unrelated individuals, often as a result of variation in the number of repeats of short base sequence
Differences between DNA fingerprinting and DNA profiling (9)
DNA profiling is more sensitive, requires smaller quantities of DNA, based on alleles, shorter time, can use several single-locus robes rather than one multi locus probe, coloured fluorescent labels rather than radioactive labels, each STR can be identified in both colour and size, produces less complex patterns and is easily interpreted.
Restriction fragment length polymorphism (RFLP)
variation that may be detected in members of a family depending on the presence or absence of a restriction enzyme cutting site within the DNA of a given gene
