Lecture 9 – Select Genetic Technologies
1953 – discovery of DNA structure
Restriction Enzymes – recognizes a specific sequence of bases anywhere within the
genome and then severs two covalent bonds (one in each strand) in the sugarphosphate
backbone at particular positions within or near that sequence recognition sequence. The
fragments generated are restriction fragments and the act of cutting is called digestion.
• Specific recognition sequences
• Can differ in length
• Result in specific cutting patterns
Fragments fit back together if they have:
• The same type of ends
• Matching sequences
Blunt Ends fragments that are cut straight
through both DNA strands right at the line of symmetry
Sticky Ends cut is displaced equally in opposite directions from the line of symmetry by
one or more bases to generate fragments with singlestranded ends
The ends can base pair with a complementary sequence form the DNA of any organism
cut by the same restriction enzyme. The longer the sequence, the less frequently there will be a cut.
Length of recognition sequences:
• 4 bp will cut every 4 = 256 bp
• 6 bp will cut every 4 = 4096 bp
• 8 bp will cut every 4 = 65.5 kb
So depending on if you want a specific average fragment length or a specific number of
fragments you will want different enzymes or different length of time for digestion
(complete vs. incomplete).
Unless otherwise stated you can assume you want complete digest the DNA has been cut
at every one of the recognition sites it contains
In incomplete digest if you don’t give the enzyme enough time to make all the cuts you
get partial cuts – this can scramble the fragments
Used to separate many different types of moleculesDNA of one length
from DNA of another, DNA from proteins, or one kind of DNA from
A solution of DNA molecules is put into wells at one end of a porous
gellike matrix (agarose).
The gel is then placed in a buffered aqueous solution and an electric
field between bare wires at either end is applied causing the negatively
charged DNA to go towards the positive pole
The gel is incubated with fluorescent DNAbinding dye called ethidium
bromide, unbound dye is washed away, and DNA can be visualized under
Small fragments migrate faster than larger ones through the pores of the
gel. So this can determine the size of the fragments.
Actual size of restriction fragments observed on gels is determined by
comparison to migration distances of known marker fragments
Genomic DNA is hundreds of thousands of fragments – because there are
so many fragments you just get a smear on the gel.
You can use this information to map when you get
your banding pattern you can use it to piece
together which band is next to which.
None of the fragments is larger than your one
biggest fragment most are smaller in the double
digest, that makes sense because there are two
enzymes making cuts
NOTE: same size doesn’t necessarily mean same
fragment. Recombinant DNA created by cutting and splicing together a vector and inserted
fragment DNA from two different organisms
Vector – selfreplicating DNA molecule that can be used to transfer DNA between host
cells and which presence can be detected
We want to keep track of the vector so we integrate the selectable marker
Plasmid – extra chromosomal DNA originally found in bacterial species
Cut the enzyme with the same restriction enzyme
used to generate the fragment of genomic DNA
and mix the digested vector and genomic DNA
together with a DNA ligase. The complementary
sticky ends will base pair.
Transformation into E. coli process by which a
cell or organism takes up a foreign DNA molecule,
changing the genetic characteristics of that cell.
The vector component of the DNA molecule:
1. Provides a receptacle for the DNA fragment of interest
2. Carries a selectable marker
3. Hijacks the cell’s biochemical machinery to amplify the recombinant molecule
4. Provides a means for distinguishing recombinant molecules from vectoronly
5. Can be trimmed away to allow purification of the amplified insert DNA
In order to propagate you transform into E. coli. You want to integrate one vector into one
Selection for Transformed Cells
Only cells containing the recombinant plasmid are able to grow and form colonies =
transformed cells. You have an ampicillin resistance as your marker so when you put the
cells on ampicillin only the cells with the
marker/vector will survive.
PCR (Polymerase Chain Reaction)
Once a sequence is known, or partially known, recover
versions of the same sequence from any source material Primers specificity
Primer length: 1825 nucleotides
Template DNA the DNA you start with (plasmid DNA, genomic DNA, restriction
fragment, PCR fragment)
Target DNA the DNA to be amplified
A specific genome region is chose and two
oligonucleotides that correspond to the two ends
of the target region are produced. These act as
primers directing the DNA polymerase.
The genomic DNA is put in a test tube with along
with the primers, a solution of four
deoxynucleotides, and Taq DNA polymerase.
Heat to 94° C for 5 min separates DNA
Lower to 5060° C for 30 sec to the primers can
Raise to 72° C for 15 minutes so that DNA
polymerase can proceed
To start the next round raise to 94° C again but o