Genetic Technologies — Lecture V

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


Sau3AI

GATC CTAG



Sau3AI

GATC CTAG

ACTGTCGATGTCGTCGTCGTAGCTGCT GATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAG CATCGATCGA


ACTGTCGATGTCGTCGTCGTAGCTGCT-3’TGACAGCTACAGCAGCAGCATCGACGACTAG-’5

5’-GATCGTAGCTAGCT 3’-CATCGATCGA

ACTGTCGATGTCGTCGTCGTAGCTGCTGATGACAGCTACAGCAGCAGCATCGACGACT

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 electrophoresisSlow: 41 bpACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

Medium: 27 bpACTGTCGATGTCGTCGTCGTAGCTGCTTGACAGCTACAGCAGCAGCATCGACGACTAG

Fast: 10 bpGATCGTAGCTAGCT CATCGATCGA F

M

S


Recessive disease allele D is cut by Sma3AI:ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

Healthy H allele is not cut:ACTGTCGATGTCGTCGTCGTAGCTGCTGAGCGTAGCTAGCTTGACAGCTACAGCAGCAGCATCGACGACTCGCATCGATCGA

HH HD DD


F

M

S

HH HD DD

A single strand of DNA will stick to its complement



60°C



95°C



60°C



1.Begin with genomic DNA2.Digest with restriction enzyme3.Separate on agarose gel4.Stain with EtBr5.Transfer to solid support6.Probe with labeled DNA


Southern blotting (named after Ed Southern)



1

2

DNA can copied by a polymerase enzyme




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 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


ACTGTCGATGTCGT


ACTGT ACTGTCGAT ACTGTCGATGT ACTGTCGATGTCGT ACTGTCGATGTCGTCGT ACTGTCGATGTCGTCGTCGT ACTGTCGATGTCGTCGTCGTAGCT ACTGTCGATGTCGTCGTCGTAGCTGCT ACTGTCGATGTCGTCGTCGTAGCTGCTGAT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT




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



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 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


Taq DNA polymerase

Thermus Aquaticus

Hot springs


• 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…


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

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Genetic Technologies — Lecture V