Download SBI 4U Genetics 6

Genetics 6:
Techniques for Producing and
Analyzing DNA
Recombinant DNA Technology
 Scientists can now construct new DNA by combining
certain genes with DNA from other areas.
 Called recombinant DNA
 Bacteria have restriction enzymes that will cut up
invading viral DNA.
 Scientists can use a special type of restriction
enzyme called restriction endonuclease because they
cleave double-stranded DNA in the middle of the
strand by recognizing a short sequence of
nucleotides called the target sequence.
Restriction Enzymes
 Are specific and predictable (same enzyme will
cleave at the same target, the same way, every time)
 Most produce a staggered cut that leaves unpaired
nucleotides at each end called sticky ends
 Sticky ends can then form base pairs with other
single-stranded regions, but only specific ends.
 If the enzyme produces blunt ends, the piece of DNA
can join with any other piece of DNA.
Steps for Making Recombinant DNA
 A restriction endonuclease is selected that can cut
both the DNA fragments that are going to be
combined.
 Each piece of DNA is reacted with the restriction
endonuclease to make the fragments.
 The two cut DNA fragments are incubated with DNA
ligase. This will seal the breaks in the DNA by forming
covalent and hydogen bonds.
Gene Cloning in Bacteria
 We can use bacteria to make many copies of
a gene for us through gene cloning.
 To do this:
 A recombinant DNA molecule made with a plasmid
mixed with the gene we want to be copied is made
 This foreign DNA is taken up by the bacteria during
transformation. Usually the cells need to be
treated with certain chemicals to make the
membranes permeable to the DNA first.
 Bacterial cells are put on a Petri dish that has
growth media on them
 Bacterial colonies with the recombinant DNA are
identified using special markers.
 Cells from the recombinant DNA colonies are
selected and grown in a liquid culture to produce a
large population.
 The recombinant DNA molecules are isolated and
purified from the bacterial cells.
 A variety of analysis techniques are used to
confirm that the correct recombinant DNA
molecule has been made.
The Polymerase Chain Reaction
(PCR)
 The process for producing large amounts of
DNA for use or analysis is called DNA
amplification.
 Polymerase Chain Reaction is a type of DNA
amplification used to amplify the DNA of
smaller segments for cloning or analysis
purposes.
 Much faster and highly specific
Steps of PCR:
 DNA sample is heated to a high temperature (95°C)
so the DNA helix denatures into single strands.
 Then it is cooled (55°C) with two nucleotide primers
that are complementary to each 3’ end. (lower temp
allows for the annealing of the primers to the DNA)
 Heated again (72°C) for Taq polymerase (type of
DNA polymerase) to work optimally by adding
nucleotides to the primers form 5’ to 3’
 Taq polymerse was isolated from heat loving bacteria.
 Repeat 1-3 (denatures, primer annealing, DNA
synthesis) several times
Gel Electrophoresis
 A technique used to separate molecules according
to their mass and charge.
 Used to separate segments of DNA.
 A gel made of agarose is submerged in a buffer.
The gel allows for the DNA to move through.
 A positive anode lies at one end and a negative
cathode at the opposite end. DNA is also negative,
so it repels and moves away from that end.
 Salts in the buffer carry the charge.
Steps in Gel Electrophoresis
 Before DNA fragments are added to the gel, a blue
dye must be added and a ethidium bromide
associates with DNA and fluoresces under UV light.
Samples of different sized fragments in solution are
then added to preformed wells at the one end of the
gel.
 Gel is placed in the buffer and a power source is
turned on. DNA fragments start to move.
 Smaller DNA fragments move more easily through
the gel and therefore travel further.
 Gel is removed from the buffer and exposed to UV
DNA Fingerprinting
 Technology used to identify individuals based
on analyzing their DNA sequence
 Use restriction enzymes to cut pieces of DNA
and then separate the DNA through gel
electrophoresis.
 To match suspect DNA with samples found at
a crime scene, bands would be the same on
the electrophoresis.
 Called restriction enzyme fragment length
polymorphism (RFLP) analysis.
Another form of DNA fingerprinting is
called short tandem repeat (STR)
profiling.
 STRs are repeating short sequences of
DNA in the genome that vary in length
between individuals.
 Numerous loci can be analyzed (the more
analyzed, the more confident the results)
Applications
Crime scene investigations
 DNA found at a crime scene is amplified by
PCR, then run through a gel
electrophoresis and compared to suspects
or victims.
Identify victims (September 11th victims)
Parental disputes
Questions
P. 291 # 1, 3, 6.
P. 295 # 7, 8, 12.