Download Genetic Investigation Technologies

Genetic Technologies
— Lecture V
• Dr. Steven J. Pittler
• WORB 658
• Office 4-6744
• Cell 612-9720
Suggested Reading: Lewis
7th Edition
Human Genetics: Concepts
and Applications
Chapter 19 Genetic
Technologies
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Amniocentesis
 In 1966 the first fetal karyotype was constructed
 Ultrasound is used to follow the needle’s movement
 Takes a few minutes and causes a feeling of
pressure
 The sampled aminotic fluid is examined for deficient,
excess, or abnormal biochemicals that could
indicated inborn errors of metabolism
 Most common chromosomal abnormality is trisomy
 Usually performed during weeks 14-16 of gestation
Amniocentesis
 Indicated when
 The risk that a fetus has a detectable condition that exceeds
the risk that the procedure will cause a miscarriage (1 in
350)
 Pregnant woman over the age of 35
 If a couple has had several spontaneous abortions
 If a couple has had a child with birth defects or a known
chromosome abnormality
Amniocentesis
 Is Indicated
 If the blood test on the pregnant woman reveals low levels of
fetal liver protein (AFP) and high levels of human chorionic
gonadotropin (hCG)
 Indicates a fetus with a small liver which may reflect a condition
caused by an extra chromosome
 Additionally
 These maternal serum marker tests may assess a third or fourth
biochemical marker as well
 The pregnancy-associated plasma protein A test (PAPP) is
detectable only during the first trimester
Chorionic Villus Sampling
• In the 10th through the 12th week of pregnancy cells can be
obtained from the chorionic villi- the structures that will
develop into the placenta
• The advantage over amniocentesis is that you do not have to
culture cells and the results can be obtained in days
• Cells from the chorionic villi descend from the fertilized ovum
and therefore they should be identical to the embryo and fetus
• Occasionally one can have a chromosomal aberration that
usually occurs either in the embryo or chronic villi
– Known as chromosomal mosaicism the karyotype of a villus cell
differs from that of an embryo cell (potential error)
Fetal Cell Sampling
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•
•
•
Safer than either of the other two procedures
Separates fetal cells from mother’s bloodstream
Technique originated in 1957
Using a device called a fluorescence-activated
cell sorter (FACS) fetal cells can be distinguished
from maternal cells
Most genetic technologies are
based on four properties of DNA
1. DNA can be cut at specific sites (motifs) by
restriction enzymes
2. Different lengths of DNA can be size-separated
by gel electrophoresis
3. A single strand of DNA will stick to its
complement (hybridization)
4. DNA can be copied by a polymerase enzyme
• DNA sequencing
• Polymerase chain reaction (PCR)
DNA can be cut at specific
sites (motifs) by an enzyme
• Restriction enzymes cut double-stranded DNA at specific
sequences (motifs)
• E.g. the enzyme Sau3AI cuts at the sequence GATC
• Most recognition sites are palindromes: e.g. the reverse
complement of GATC is GATC
• Restriction enzymes evolved as defense against foreign
DNA
Sau3AI
GATC
CTAG
DNA can be cut at specific
sites (motifs) by an enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
DNA can be cut at specific
sites (motifs) by an enzyme
Sau3AI
GATC ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
CTAG TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
DNA can be cut at specific
sites (motifs) by an enzyme
Sau3AI
ACTGTCGATGTCGTCGTCGTAGCTGCT GATC
GATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAG
CTAG CATCGATCGA
DNA can be cut at specific
sites (motifs) by an enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCT-3’
TGACAGCTACAGCAGCAGCATCGACGACTAG-’5
5’-GATCGTAGCTAGCT
3’-CATCGATCGA
ACTGTCGATGTCGTCGTCGTAGCTGCTGA
TGACAGCTACAGCAGCAGCATCGACGACT
TCGTAGCTAGCT
AGCATCGATCGA
Different lengths of DNA can be
separated by gel electrophoresis
• DNA is negatively charged and will move through a gel matrix
towards a positive electrode
• Shorter lengths move faster
Different lengths of DNA can be
separated by gel electrophoresis
Slow: 41 bp
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
Medium: 27 bp
ACTGTCGATGTCGTCGTCGTAGCTGCT
TGACAGCTACAGCAGCAGCATCGACGACTAG
S
M
Fast: 10 bp
GATCGTAGCTAGCT
CATCGATCGA
F
Different lengths of DNA can be
separated by gel electrophoresis
Recessive disease allele D is cut by Sma3AI:
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
Healthy H allele is not cut:
ACTGTCGATGTCGTCGTCGTAGCTGCTGAGCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTCGCATCGATCGA
HH
HD
DD
Different lengths of DNA can be
separated by gel electrophoresis
HH
S
M
F
HD
DD
A single strand of DNA will
stick to its complement
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A single strand of DNA will
stick to its 60°C
complement
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A single strand of DNA will
stick to its 95°C
complement
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A single strand of DNA will
stick to its 60°C
complement
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A single strand of DNA will
stick to its complement
1. Begin with genomic DNA
2. Digest with restriction enzyme
3. Separate on agarose gel
4. Stain with EtBr
5. Transfer to solid support
6. Probe with labeled DNA
A single strand of DNA will
stick to its complement
Southern blotting (named after Ed Southern)
A single strand of DNA will
stick to its complement
A single strand of DNA will
stick to its complement
1
2
DNA can copied by a
polymerase enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
DNA can copied by a polymerase enzyme
G
A
G
A
C
C
DNA
polymerase
T
A
G
T
G
G
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A
A
G
T
C
A
C
C
A
G
A
C
T
T
A
G
T
G
DNA can copied by a polymerase enzyme
G
A
G
A
C
C
DNA
polymerase
T
A
G
T
G
G
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A
A
G
T
C
A
C
C
A
G
A
C
T
T
A
G
T
G
DNA can copied by a polymerase enzyme
ACTGTCGATGTCGT
DNA can copied by a polymerase enzyme
ACTGT
ACTGTCGAT
ACTGTCGATGT
ACTGTCGATGTCGT
ACTGTCGATGTCGTCGT
ACTGTCGATGTCGTCGTCGT
ACTGTCGATGTCGTCGTCGTAGCT
ACTGTCGATGTCGTCGTCGTAGCTGCT
ACTGTCGATGTCGTCGTCGTAGCTGCTGAT
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGT
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCT
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT
DNA can copied by a polymerase enzyme
DNA can copied by a polymerase enzyme
DNA can copied by a polymerase enzyme
Polymerase chain reaction (PCR)
• A method for producing large (and
therefore analysable) quantities of a
specific region of DNA from tiny quantities
• PCR works by doubling the quantity of
the target sequence through repeated
cycles of separation and synthesis of
DNA strands
DNA can copied by a polymerase enzyme
DNA can copied by a polymerase enzyme
G
A
C
T
A thermal cycler (PCR machine)
Forward
Reverse G, A, C, T
primer Heat resistant DNA DNA template
primer
bases
polymerase
DNA can copied by a polymerase enzyme
DNA can copied by a polymerase enzyme
Increase in DNA quantity in PCR
1.0E+11
Quantity of DNA relative to initial sample
1.0E+10
1.0E+09
Theory
Practice
1.0E+08
1.0E+07
1.0E+06
1.0E+05
1.0E+04
1.0E+03
1.0E+02
1.0E+01
1.0E+00
0
5
10
15
20
Cycle number
25
30
35
DNA can copied by a polymerase enzyme
Taq DNA
polymerase
Thermus
Aquaticus
Hot springs
DNA can copied by a polymerase enzyme
In the words of its inventor, Kary Mullis…
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PCR can generate 100 billion copies from a single DNA molecule in an afternoon
PCR is easy to execute
The DNA sample can be pure, or it can be a minute part of an extremely complex
mixture of biological materials
The DNA may come from
– a hospital tissue specimen
– a single human hair
– a drop of dried blood at the scene of a crime
– the tissues of a mummified brain
– a 40,000-year-old wooly mammoth frozen in a glacier.
DNA can copied by a polymerase enzyme
Microarrays
Gene expression
• Transcription:
– DNA gene → mRNA
– in nucleus
• Translation:
– mRNA → protein
– in cytoplasm
• Microarrays use mRNA
as a marker of gene
expression
Nucleus
Cytoplasm
What are microarrays?
• A microarray is a DNA “chip” which holds 1000s of
different DNA sequences
• Each DNA sequence might represent a different gene
• Microarrays are useful for measuring differences in gene
expression between two cell types
• They can also be used to study chromosomal
aberrations in cancer cells
Principles behind microarray analysis
• Almost every cell in the body contains all
~35,000 genes
• Only a fraction is switched on (expressed)
at any time in any cell type
• Gene expression involves the production
of specific messenger RNA (mRNA)
• Presence and quantity of mRNA can be
detected by hybridization to known RNA
(or DNA) sequences
What can microarray analysis tell us?
• Which genes are involved in
– disease?
– drug response?
• Which genes are
– switched off/underexpressed?
– switched on/overexpressed?
Microarray analysis: probe preparation
Microarray analysis: target preparation
50 x 50 array =
2500 genes
sampled
Microarrays can be used to diagnose
and stage tumours, and to find genes
involved in tumorigenesis
• Copy number changes are common in tumours
• Loss or duplication of a gene can be a critical stage in tumour
development
Chromosome 1
2
3
4
5
6
7
8
9
10
11
12
13
14 15 16 17 18 19 202122
BMC Cancer 2006, 6:96
Problems of microarray analysis
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Gene expression ≠ mRNA concentration
Easy to do, difficult to interpret
Standardization between labs
Lots of noise, lots of genes (parameters)
– e.g. p = 10,000
• low sample size
– e.g. n = 3