Download The Body in Motion

Lecture ONE:
Foundation Course Genetics
Tools of Human Molecular Genetics I
Discuss the following:
• Genetic engineering
• Molecular Genetics
• Genetic Screening
• Recombinant DNA
• Stem cells
Biology, Seventh Edition
CHAPTER 14 DNA Technologies
Recombinant DNA methods
• Restriction enzymes
–Enzymes from bacteria
–Used to cut DNA molecules in specific places
• cut DNA is placed in a Vector carrier
• DNA ligase joins cut pieces of DNA
• Transformation: uptake of foreign DNA
into cells
3
Copyright © 2005 Brooks/Cole — Thomson Learning
Cloning Vectors
Vector
Maximum
Insert size
Approx. No. of clones required
in library
plasmid
10 kb
10 x 105
lambda
20 kb
5 x 105
cosmid
45 kb
2 x 105
YAC
1 Mb
104
BAC
> 500 kb
5 x 104
Biology, Seventh Edition
CHAPTER 14 DNA Technologies
Plasmids
5
Copyright © 2005 Brooks/Cole — Thomson Learning
Biology, Seventh Edition
CHAPTER 14 DNA Technologies
Cutting DNA with a restriction enzyme
6
Copyright © 2005 Brooks/Cole — Thomson Learning
Restriction Enzymes fig 14.2
• Restriction enzymes are molecular
scissors which cut DNA at specific
base sequences.
E.g. HindIII and EcoR1 are restriction
enzymes
• Hind III cuts DNA at the recognition
sequence 5’-AAGCTT-3’
• EcoR1 cuts DNA at the recognition
sequence 5’- GAATTC-3’
Restriction Enzymes: (Fig 14-1)
• Many of the sites in DNA which
restriction enzymes recognize are
PALINDROMIC
• Palindromic: means that the sequence
reads the same 5’-3’ in both directions
Biology, Seventh Edition
CHAPTER 14 DNA Technologies
Cutting DNA with a restriction enzyme
9
Copyright © 2005 Brooks/Cole — Thomson Learning
Biology, Seventh Edition
CHAPTER 14 DNA Technologies
• Splicing foreign DNA into a vector
• Foreign DNA and plasmid DNA cut with
same restriction enzyme
• Produces linear molecules with
complementary single-stranded ends
• Recombinant DNA created by mixing so
sticky ends pair
• DNA ligase forms covalent bonds, linking
the two fragments
10
Copyright © 2005 Brooks/Cole — Thomson Learning
Biology, Seventh Edition
CHAPTER 14 DNA Technologies
Producing a
genomic or
chromosome
library
11
Copyright © 2005 Brooks/Cole — Thomson Learning
• Genomic library
• Collection of DNA fragments that
represent all the DNA in the genome
• Chromosome library
• All the DNA fragments in that specific
chromosome
• cDNA library
• Produced using reverse transcriptase
• Makes DNA copies of mature mRNA
Biology, Seventh Edition
CHAPTER 14 DNA Technologies
Cloning DNA:
• The recombinant DNA can be put back into
bacteria and the bacteria allowed to grow
• This will produce many genetically identical
copies of the piece of DNA. This is called
cloning
• A clone is a genetically identical individual or
cell
13
Copyright © 2005 Brooks/Cole — Thomson Learning
Biology, Seventh Edition
CHAPTER 14 DNA Technologies
• Genetic probes
• Segments of single-stranded DNA that
can hybridize to complementary base
sequences in target gene
• Southern blot technique
14
Copyright © 2005 Brooks/Cole — Thomson Learning
Southern Northern, and Western
Blotting
•Southern Blotting: DNA
•Northern Blotting: RNA
•Western Blotting : Protein
Biology, Seventh Edition
CHAPTER 14 DNA Technologies
Using a genetic
probe to find
bacterial cells
with a specific
recombinant
DNA molecule
16
Copyright © 2005 Brooks/Cole — Thomson Learning
Biology, Seventh Edition
CHAPTER 14 DNA Technologies
• Amplifying DNA in vitro by PCR
–Small amount of double-stranded DNA
–DNA precursors
–Specific nucleic acid primers
–Taq DNA polymerase
• DNA is denatured
• Primers attach to primer-binding site on
each DNA strand
• Each strand acts as template for DNA
synthesis
17
Copyright © 2005 Brooks/Cole — Thomson Learning
Biology, Seventh Edition
CHAPTER 14 DNA Technologies
Amplification of DNA by PCR
18
Copyright © 2005 Brooks/Cole — Thomson Learning
Types of PCR I
RT PCR
Reverses transcriptase PCR. DNA is amplified
from mRNA.
QRT PCR
Quantitative Real Time PCR
the amplified product is linked to a fluorescent
reporter molecule, the fluorescence is measured
at each cycle. This allows the amplification to be
monitored to optimize the efficiency of
amplification.
Types of PCR II
Multiplex PCR
two or more sets of primers are used in
the same reaction mix.
Nested PCR
(also nested RT-PCR)
There are two amplifications made: the:
The first amplifying a large product
which, in the second amplification is
used as the template.
Gel Electrophoresis
• A method of separation
pieces of DNA
• Horizontal gel (Agarose)
• Vertical (Polyacrylamide)
• Many variations on the methods exist
The DNA is negatively charged and
migrates towards the positive pole
The smallest fragments move the fastest
through the gel and therefore move the
furthest
The largest fragments move the least
By comparing the fragments in the sample
with standards of known size
the size of the sample fragments can be
estimated
Links to virtual labs
• DNA extraction
http://learn.genetics.utah.edu/units/
biotech/extraction/
• Making a gel
http://learn.genetics.utah.edu/units/
biotech/gel
Stem Cells are undifferentiated cells
Type of Stem Cell
Early Embryonic
Blastocyst Embryonic
Fetal
Umbilical
Adult
Developmental
Potential
Totipotent
Pluripotent
Pluripotent
Multipotent
Multipotent
http://learn.genetics.utah.edu/units/stemcells
Biology, Seventh Edition
CHAPTER 14 DNA Technologies
Possible therapeutic uses of stem cells
• Treatment of disease
• Diabetes
• Parkinsons disease
• Treatment of spinal injury
• Culture of differentiated
tissues
26
Copyright © 2005 Brooks/Cole — Thomson Learning