Transcript Chapter 13: The Molecular Basis of Inheritance

Chapter 13: The
Molecular Basis of
Inheritance
Concept 13.4: Understanding DNA structure and replication makes
genetic engineering possible
Overview
●
Knowledge that two strands of DNA are complements of each other acts as a fundamental in
modern genetics and genetic research
○ It is the basis upon nucleic acid hybridization, that the base pairing of one strand of nucleic
acid to a complementary sequence on another strand, is built
○ Nucleic acid hybridization is the foundation for most techniques used for genetic engineering,
the direct manipulation of of genes for practical purposes
Genetic Engineering has drastically changed the fields of medicine, forensics, agriculture, and simple
biological research
DNA Cloning: Making Multiple Copies of a Gene or
Other DNA Segment
◉ Naturally occurring DNA molecules are very long, and a single molecule usually carries
many genes.
◉ To work directly with specific genes, scientists have developed methods for preparing
well-defined segments of DNA in multiple identical copies, a process called DNA cloning.
○ One common approach uses bacteria, most often E. coli, which has large circular
molecules of DNA.
■ To clone the DNA, researchers first obtain a plasmid, a small circular DNA
molecule that replicates separately from the bacterial chromosome, and insert
DNA from another source into it, resulting in recombinant DNA.
■ The new plasmid is then returned to a bacterial cell, creating recombinant
bacterium.
■ This single cell reproduces through repeated cell divisions to form a population
of genetically identical cells.
Applications of Gene Cloning
◉ Gene cloning is useful for two basic purposes- to amplify, or make copies of a
gene, and to produce a protein product.
○ Organisms can be given new, metabolic traits, such as pest resistance.
○ Proteins with medical uses can be harvested in large quantities from
cultures of bacteria carrying the cloned gene from the protein.
Using Restriction Enzymes to Make Recombinant DNA
◉ Overview
○ Genetic cloning and engineering uses restriction enzymes to recognize and cut
DNA at specific locations called restriction sites.
■ This was discovered while researching bacteria, which protects it by
cutting foreign DNA, but it protects itself from its own enzymes by adding
methyl groups to adenines and cytosines in recognized sequences.
○ When DNA is cut, it creates a set of segments called restriction fragments.
■ Fragments are identical when cut with the same enzyme.
● Most restriction sites are symmetric
(nucleotides are the same on each strand,
same in 5’ → 3’)
● Most commonly used is
4’ → 8’, occurs many times
Over DNA molecule
● This restriction enzyme recognizes 6 base
pair sequences making staggered cuts in
the sugar phosphate backbones
● Results in sticky ends, any fragments with
complementary ends may base- pair.
○ the resulting ligated product will be
recombinant DNA if the fragments
come from different molecules
●
●
●
●
When DNA is cut, it creates a set of segments
called restriction fragments. Fragments are
identical if cut with the same enzyme.
Seeing these requires electrophoresis- polymer
separates nucleic acids by size, electric charge
and other physical properties in the electric
field.
Sugar phosphate backbones in DNA strands
result in double stranded fragments, which
have a single stranded end, or sticky end
forming temporary hydrogen bonds with
molecules cut by the same enzyme.
In gene cloning, DNA molecules that are to be
joined together are a cloning vector- a DNA
molecule able to carry foreign DNA to a host
cell for replication, often a bacterial plasmid
with one restriction site recognized by its
specific restriction enzyme
Amplifying DNA in Vitro: The Polymerase Chain Reaction
(PCR) and Its Use in Cloning
◉ When scientists need to analyze DNA with only a small sample size, they use
polymerase chain reaction (PCR) to amplify and copy genetic material
○ PCR has the capability to make millions (or even billions) of copies of small
sections of DNA
Number of copies equals 2^n, were n is the number of cycles
A target sequence can make up as little as 0.001% of the total DNA in a
sample
○
PCR cannot replace gene cloning because of its error rate as samples
become larger
○
PCR has been used to amplify DNA from 40,000 year-old frozen woolly
mammoth, small amounts of DNA at crime scenes, and single embryonic
cells for unique and fast prenatal treatment for genetic disorders
Essentially, there are three parts...
Denaturation
Annhelling
The DNA is heated to
The DNA is cooled to allow
separate the two strands for short-sequence primers
to attach to anneal (base
pair) with its complementary
sequence. The primers
isolate the target sequence
for amplification.
Extention
Temperature is raised
once again to allow Taq
polymerase to attach to
each priming site and
extend (synthesize) a
new DNA strand.
The Secret To Polymerase
Chain Reaction
◉ If a normal DNA polymerase were used, both the protein and DNA
would be denatured in the heating at the beginning of the cycle
○ This means the protein would have to be replaced after every
cycle
○ The key to PCR being an automatic procedure is a the discovery
of a heat-stable polymerase called Taq polymerase
■ Named after the bacteria Thermus aquaticus which lives in
hot springs
■ This bacteria can live in temperatures up to 95 degrees
celsius
DNA Sequencing
◉ DNA sequencing- the process of exploiting the principle of complementary base pairing to
determine the gene’s complete nucleotide sequence
◉ Recent developments have lead to sequencing techniques that are rapid and inexpensive.
○
Now, a single template strand can be immobilized and DNA polymerase, among other
reagents, are added to allow sequencing by synthesis of the complementary strand, one
nucleotide at a time.
○
Electronic monitors are used to identify which of the four nucleotides are being added,
determining the sequence.
Bibliography
Reece, Jane B, and Neil A.
Campbell. Campbell Biology.
Boston: Benjamin Cummings /
Pearson, 2011. Print.
“
Thanks!
Any questions?