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Gene Technology
Making DNA Fragments
Gene technology ​all the techniques that can be used to study genes and their function
is
It is used all about studying genes
It uses DNA fragments to do this
There are three ways to get a copy of the DNA fragment containing the gene you’re interested
in
Reverse Transcriptase
Many cells only contain two copies of each gene
This makes it difficult to obtain a DNA fragment containing the target gene
They can contain many mRNA molecules complementary to the gene
mRNA is often easier to obtain
The mRNA molecules can be used as templates to make lots of DNA
The enzyme reverse transcriptase makes DNA from an RNA template
The DNA produced is called complementary DNA, or cDNA
To do this, mRNA is isolated from cells
It is then mixed with free DNA nucleotides and reverse transcriptase
The reverse transcriptase uses the mRNA as a template to synthesize a new strand of cDNA
Restriction Endonuclease Enzymes
Some sections of DNA have palindromic sequences of nucleotides
These sequences consist of antiparallel base pairs
Antiparallel base pairs ​ base pairs that read the same in opposite directions
are
Restriction endonucleases are enzymes that recognise specific palindromic sequences and
digest the DNA at these places
Palindromic sequences are known as recognition sequences
Different restriction endonucleases cut at different specific recognition sequences

The shape of the recognition sequence is complementary to an enzyme’s active site
If recognition sequences are present at either side of the DNA fragment you want, you can use
restriction endonucleases to separate it from the rest of the DNA
The DNA sample is incubated with the specific restriction endonuclease
This cuts the DNA fragment out via a hydrolysis reaction
Sometimes the cut leaves sticky ends
Sticky ends ​ small tails of unpaired bases at each end of the fragment
are
Sticky ends can be used to bind or anneal the DNA fragment to another piece of DNA that has
sticky ends with complementary sequences
Polymerase Chain Reaction
A polymerase chain reaction can be used to make millions of copies of a fragment of DNA
It has several stages and is repeated again and again
This ensures lots of copies are made
A reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA
polymerase
Primers ​ short pieces of DNA that are complementary to the bases at the start of the
are
fragment you want
DNA polymerase ​an enzyme that creates new DNA strands
is
The DNA mixture is heated to 95O​
​
C
This breaks up the hydrogen bonds between the two strands of DNA
The mixture is then cooled to between 50 and 65O​
​
C
This allows the primers to anneal to the strands
The reaction mixture is heated to 72O​
​
C
This allows DNA polymerase to work
The DNA polymerase lines up free DNA nucleotides alongside each template strand
Specific base pairing means new complementary strands are formed
Two new copies of the fragment of DNA are formed

One cycle of polymerase chain reaction is complete
The cycle starts again
The mixture is heated to 95O​ and all four strands are used as templates
​
C,
These strands include the two original and two new
Each cycle doubles the amount of DNA
For example:
First Cycle - 2 x 2 = 4 DNA fragments
Second Cycle - 4 x 2 = 8 DNA fragments
Fifth Cycle - 32 x 2 = 64 DNA fragments

Gene Cloning
Gene cloning is about making identical copies of a gene
It can be done using two techniques:
In vitro ​
cloning is where the gene copies are made outside of a living organism using
polymerase chain reaction
In vivo ​
cloning is where the gene copies are made within a living organism
As the organisms grows and divides, it replicates its DNA
This creates multiple copies of the gene
In vivo Cloning
1
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The vector with the recombinant DNA is used to transfer the gene into host cells
If a plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its
DNA
If a bacteriophage vector is used, the bacteriophage will infect the host bacterium by injecting
its DNA into it
The phage DNA which contains the target gene then integrates into the bacterial DNA
Host cells that take up the vectors containing the gene of interest are said to be transformed
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