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
FETAL
TESTING
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
• 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)
• 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
DNA can be cut at specific sites (motifs) by an enzyme
Sau3AI
GATC CTAG
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
DNA can be cut at specific sites (motifs) by an enzyme
Sau3AI
GATC CTAG
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
DNA can be cut at specific sites (motifs) by an enzyme
Sau3AI
GATC CTAG
ACTGTCGATGTCGTCGTCGTAGCTGCT GATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAG CATCGATCGA
DNA can be cut at specific sites (motifs) by an enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCT-3’TGACAGCTACAGCAGCAGCATCGACGACTAG-’5
5’-GATCGTAGCTAGCT 3’-CATCGATCGA
ACTGTCGATGTCGTCGTCGTAGCTGCTGATGACAGCTACAGCAGCAGCATCGACGACT
TCGTAGCTAGCT AGCATCGATCGA
DNA can be cut at specific sites (motifs) by an enzyme
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 electrophoresisSlow: 41 bpACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
Medium: 27 bpACTGTCGATGTCGTCGTCGTAGCTGCTTGACAGCTACAGCAGCAGCATCGACGACTAG
Fast: 10 bpGATCGTAGCTAGCT CATCGATCGA F
M
S
Different lengths of DNA can be separated by gel electrophoresis
Recessive disease allele D is cut by Sma3AI:ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
Healthy H allele is not cut:ACTGTCGATGTCGTCGTCGTAGCTGCTGAGCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTCGCATCGATCGA
HH HD DD
Different lengths of DNA can be separated by gel electrophoresis
F
M
S
HH HD DD
A single strand of DNA will stick to its complement
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A single strand of DNA will stick to its complement
60°C
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A single strand of DNA will stick to its complement
95°C
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A single strand of DNA will stick to its complement
60°C
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
A single strand of DNA will stick to its complement
1.Begin with genomic DNA2.Digest with restriction enzyme3.Separate on agarose gel4.Stain with EtBr5.Transfer to solid support6.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
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
DNA can copied by a polymerase enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
DNA polymerase
C
CC
CCC
G
G
G
G
G
GG
G
G
T T
T
T
A
T
T
A
A
A
AA
A
A
A
DNA can copied by a polymerase enzyme
ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA
DNA polymerase
C
CC
CCC
G
G
G
G
G
GG
G
G
T T
T
T
A
T
T
A
A
A
AA
A
A
A
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
A
C
T
G
DNA templateHeat resistant DNA polymerase
G, A, C, T bases
Forward primer Reverse
primer
A thermal cycler (PCR machine)
DNA can copied by a polymerase enzyme
DNA can copied by a polymerase enzymeIncrease in DNA quantity in PCR
1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
1.0E+08
1.0E+09
1.0E+10
1.0E+11
0 5 10 15 20 25 30 35Cycle number
Qua
ntity
of D
NA
rela
tive
to in
itial
sam
ple Theory
Practice
DNA can copied by a polymerase enzyme
Taq DNA polymerase
Thermus Aquaticus
Hot springs
DNA can copied by a polymerase enzyme
• 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.
In the words of its inventor, Kary Mullis…
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• 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