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Studying and Manipulating
Genes

Cloning DNA

 Researchers cut up DNA from different sources,
then paste the resulting fragments together
 Cloning vectors can carry foreign DNA into
host cells

Cut and Paste
• Researchers use restriction enzymes to cut up DNA,
then bond the fragments together using DNA ligase
• Fragments with complementary tails (“sticky ends”)
stick together when their matching tails base-pair
 Single-stranded DNA tails produced by the same
restriction enzyme base-pair together
• DNA ligase bonds “sticky ends” together

• restriction enzyme
• Bacterial enzyme used to cut specific nucleotide
sequences in DNA

 Recombinant DNA
• Composed of DNA from two or more organisms

Making Recombinant DNA

restriction
enzyme (cut)

A A restriction
enzyme recognizes
a specific base sequence in
DNA
...


mix

B Researchers use
restriction enzymes to
cut DNA from different
sources into fragments
...


DNA ligase
(paste)

C Matching sticky
ends of different
fragments base-pair
with each other,
regardless of the
source of the DNA
...
Molecules
of recombinant DNA
are the result
...
16-2, p
...
85 kb

Sal l

Acc l
Xho l
Xba l
Bst XI

Sac l
A

B

Not l

Fig
...
3, p
...


B The same
enzyme cuts the
same sequence
in plasmid DNA
...


D The plasmid DNA
also has sticky ends
...
The
sticky ends
of different
fragments
that basepair are
bonded by
DNA ligase
...

These plasmids are
introduced into host
cells, which divide
to form clones
...

The cells divide repeatedly and form colonies—clusters
of millions of genetically identical daughter cells
...


C The paper is soaked in a solution that ruptures the
cells and releases their DNA
...


D A probe is added to the liquid bathing the paper
...


E The bound probe makes a spot
...
The position of the spot on the film
is compared to the positions of all the original bacterial
colonies
...

Fig
...
244

Big-Time Amplification: PCR
• The polymerase chain reaction (PCR) uses primers
and heat-resistant DNA polymerase to mass-produce
a particular section of DNA without having to clone it in
living cells
• polymerase chain reaction (PCR)
• Method that rapidly generates many copies of a
specific section of DNA

• primer
• Short, single strand of DNA designed to hybridize with
a DNA fragment

PCR

 DNA to be copied is mixed with DNA
polymerase, nucleotides and primers that basepair with certain DNA sequences
 Cycles of high and low temperatures break and
reform hydrogen bonds between DNA strands,
doubling the amount of DNA in each cycle

PCR

A DNA template (purple) is mixed with
primers (red), free nucleotides, and
heat-tolerant Taq DNA polymerase
...
When it is cooled, some primers
hydrogen-bond to the template DNA
...
The first round of PCR is now
complete
...

When the mixture is cooled, some of the
primers hydrogenbond to the DNA
...
The second round of PCR is complete
...
After 30 rounds, the mixture contains
huge numbers of DNA fragments, all copies of
the template DNA
...
16-6, p
...

The mixture also includes
the four dideoxynucleotides
labeled with four different
colored pigments
...

Synthesis of each new
strand stops when a dideoxynucleotideis added
...

E A computer detects
and records the color of
each band on the gel
...


D An electrophoresis gel separates the fragments
into bands according
to length
...


DNA Fingerprinting

 One individual can be distinguished from all
others on the basis of DNA fingerprints

DNA Fingerprints
 DNA fingerprint
• A unique array of DNA sequences used to
identify individuals

 Short tandem repeats (STRs)
• Many copies of the same 2- to 10-base-pair
sequences in a series along a chromosome
• Types and numbers of STRs vary greatly
among individuals

Creating DNA Fingerprints
 PCR is used to amplify DNA from regions of
several chromosomes that have STRs
 Electrophoresis is used to separate the
fragments and create a unique DNA fingerprint
 DNA fingerprints have many applications
• Legal cases, forensics, population studies

DNA Fingerprints: A Forensics Case



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