U3: Molecular Genetics SBI4U

L8: Restriction Enzymes and Gene Cloning

Recombinant DNA Technology
- DNA that has been processed in a lab, combining fragments of DNA
from more than one source (i.e., different species)
- Restriction enzymes, only found in prokaryotes, cut DNA at specific
sites called restriction sites
- EcoRI = 5’ G / A A T T C – 3’

Two Useful Characteristics:

(1) Specificity – DNA cuts are specific & predictable
(2) Staggered Cuts – also known as ‘sticky ends’; allow unpaired
nucleotides at each end to base pair with another strand

Steps for producing Recombinant DNA Molecule

- Restriction enzyme cuts the DNA at restriction
sites
- DNA from another source is joined to the
original sticky ends using DNA ligase

Sticky Ends Restriction enzyme cuts the DNA

DNA from another

source cut with some
restriction enzymes
(same sticky ends)

DNA ligase joins

fragments
U3: Molecular Genetics SBI4U
L8: Restriction Enzymes and Gene Cloning
PRACTICE
Look at the sequence below. If you add a restriction enzyme (EcoRI = G/AATTC) to the uncut DNA, how many DNA
fragments will result? Circle/highlight the fragments

9 bp 9 bp 9 bp 7 bp
4 9, 9, 9 and 7

22 bp 9 bp 7 bp
3 22, 9, and 7

2 22 and 20

Restriction Enzymes, and Gene Cloning using Bacteria

- Gene cloning involves making many copies of a
gene to study or make proteins from
- Bacteria are good hosts because they multiply
quickly, are east to maintain, and are inexpensive
- Bacteria replicate = target gene replicates
- Either gene or protein from gene can be used in
different applications (Step #4)
U3: Molecular Genetics SBI4U
L8: Restriction Enzymes and Gene Cloning
The Polymerase Chain Reaction (PCR)
- Method for amplifying (making many copies) of specific DNA region from a small quantity (i.e., nose swab,
blood smear, hair, bones, etc.)
- A machine that can thermoregulate quickly to make many copies of a strand of DNA

Steps in PCR

Step 1 - DNA sample is heated

approximately 95°C causing DNA to
denature (split)

Step 2 – DNA is cooled to approximately

55°C, allowing DNA primers to anneal to the
ends of the DNA to be amplified

Step 3 – DNA heated back up to

approximately 72°C, which is the optimal
temperature of Taq polymerase (found in
heat-loving bacteria)

Step 4 – Repeat steps #1-3 until desired

amount of copies are made

PRACTICE
What are the necessary ingredients to go inside the PCR for gene
amplification to happen correctly?
DNA primers, Taq polymerase, a DNA sample, and may nucleotides
are required in PCR gene amplification.

A PCR in your lab develops a malfunction where it only heats up to 72 oC.

What effect would this have on the process, and why?
If the PCR is only able to heat up to 72°C, this may affect the amplification process, as the PCR typically heats up to
95°C so that the DNA can denature. Heating up the DNA disrupts the hydrogen bonds that link base pairs, allowing

the DNA to unwind. If the PCR is unable to reach the ideal temperature, the process may take longer or not work.
U3: Molecular Genetics SBI4U
L8: Restriction Enzymes and Gene Cloning
Check Your Understanding
1. As a scientist, would you rather use a restriction enzyme that produces “sticky ends” with overhanging
nucleotides, or blunt, straight cuts with no overhang? Explain your reasoning.
As a scientist, I would rather use a restriction enzyme that produces “sticky ends” because it has more useful
characteristics. Using restriction enzymes that produce sticky ends leave staggered cuts, allowing unpaired

nucleotides to base pair with another strand.

- Sticky ends are easier to use – easy to recombine with other sticky ends
- Blunt ends are better to combine 2 genes with less specificity – does not require certain nucleotides

2. Complete the following table

G AATTC Sticky ends

+
CTTAA G

G GATCC
+ Sticky ends
CCTAG G

A AGCTT
+ Sticky ends
TTCGA A

TC + GA Blunt ends
AG CT

C CCGGG
+ Sticky ends
GGGCC C

C + TGCAG Sticky ends

GACGT C

GG + C C
C C Blunt ends
GG

(a) Use BamHI to cut the following sequence (b) Use HindIII to cut the following sequence

(c) Use EcoRI to cur the following sequence (d) Use Sma1 to cut the following sequence

(e) Use Taq1 to cut the following sequence (f) Use Pst1 to cut the following sequence
 U3: Molecular Genetics SBI4U
L8: Restriction Enzymes and Gene Cloning
3. The following DNA sequence is from a virus that is dangerous, and scientists want to use a restriction enzyme to
cut the virus’ DNA into bits. They do not need sticky ends and they won’t combine it with any other DNA. Choose
one of your restriction enzymes from above to cut the DNA below. How many segments are you left with?

Using the Hae111 restriction enzyme, I cut the DNA sequence, leaving me with four segments.

4. Explain the role of each of the following in the PCR

a. DNA primer
DNA primers initiate the PCR reaction by binding to and annealing the ends of the DNA to be amplified

b. Choice of Taq polymerase

Taq polymerase adds in the new DNA bases, building a new complementary strand (synthesis of daughter strand)

c. Cycling through different temperatures

Allows the strands in the DNA sample to denature and anneal to make more copies.

5. How has PCR technology revolutionized genetic testing? What are some possible advantages/disadvantages for
society?
PCR technology allows segments of DNA to reproduce many millions of copies of the specific DNA sequence from

a small sample. PCR testing is used to analyze crime scenes, gene cloning, testing for viruses, and much more. An
advantage of PCR technology includes flexibility, its inexpensive, allows for increased specificity, it only requires a

small sample and is time efficient. Disadvantages include lower specificity rates compared to cultures, may have
inaccurate results, cannot be used to amplify unknown targets, and are sensitive to contamination.

Another consideration that should be made as technology advances is the ethical concerns surrounding gene

cloning. Gene cloning, specifically for humans, is considered controversial because of the high likelihood of loss of
life, the possibility of eugenics, and some consider it to be “unnatural.”