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682 Chapter 20 NEL

DNA Ligase

To create recombinant DNA, pieces of DNA from two sources must be joined together.

Using restriction enzymes and methylases, molecular geneticists can engineer fragments

of DNA that contain the specific nucleotide sequences they want. These segments of

DNA are then joined together by DNA ligase.If two fragments have been generated using

the same restriction enzyme, they will be attracted to each other at their complementary

sticky ends. Hydrogen bonds will form between the complementary base pairs. DNA ligase

then joins the strands of DNA together (Figure 6).

AAGCAG TCGACATGCA

TTCGTCAGCT G TACGT

3 


5 

5 

DNA ligase

AAGCAGAAT T CATA

TTCGTCAGCTG TAT

3 


5 

AAGCAGTCGACATGCA

TTCGTCAGCTG TACGT

3 


5 

3 

5 


3 

5 


3 


HindIII fragment

(c) EcoRI fragment

(b)

(a)

DNA ligase

Figure 6

DNA ligase is able to join

complementary sticky ends

produced by the same restriction

enzyme via a condensation reaction.

(a)Complementary sticky ends

produced by HindIII

(b)Hydrogen bonds form between

complementary bases. DNA

ligase creates bonds between

nucleotides in the DNA

backbones.

(c)If fragments are not

complementary, then hydrogen

bonds will not form.

TaqDNA Polymerase and the Polymerase Chain Reaction

In 1985, American scientist Kary Mullis invented a biotechnology technique called the

polymerase chain reaction(PCR). PCR allows scientists to make billions of copies of

pieces of DNA from extremely small quantities of DNA. The reaction depends on the spe-

cial property ofTa qpolymerase. In nature,Ta qDNA polymerase is found in the bacterium

Thermos aquaticus, which lives at extremely high temperatures. Like all the DNA poly-

merases,Ta qDNA polymerase synthesizes DNA during replication. As you have learned

previously, enzymes have an optimum temperature range in which they function. One

adaptation that allows Thermos aquaticusto survive at high temperatures is that its DNA

polymerase is stable at much higher temperatures than DNA polymerases from other

organisms. Mullis used the heat-stable property ofTa qpolymerase in his PCR

technique.

To prepare for PCR, the following materials are placed together in a small tube:Ta q

polymerase, the DNA to be copied, a large quantity of the four deoxynucleotides (A, T,

G, and C), and short primers. The tube is then inserted into a PCR machine. PCR involves

four simple steps (Figure 7).

polymerase chain reaction(PCR)

a technique for amplifying a DNA

sequence by repeated cycles of

strand separation and replication

DID YOU KNOW??

Eukaryotic Methylation

Methylases in eukaryotes are

connected with the control of

transcription. In addition,

approximately 2 % of mammalian

ribosomal RNA is methylated after it

is transcribed.

initial DNA sample

single-stranded

DNA templates

heat 4.

cool primer

complementary DNA copy

Figure 7

Steps in the PCR

1. The mixture is heated to a temperature high enough to break the hydrogen bonds in the
   double helix of the DNA and separate the strands. This forms single-stranded DNA
   templates.


2. The mixture is cooled, and the primers form hydrogen bonds with the DNA templates.


3. Taqpolymerase synthesizes a new stand of DNA from the DNA template by
   complementary base pairing, starting at each primer.


4. The cycle of heating and cooling is repeated many times.