green with envy?? jelly fish “gfp” transformed vertebrates
TRANSCRIPT
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Green with envy??Jelly fish “GFP”
Transformed vertebrates
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DNA Manipulation, Gene Cloning and Karyotyping
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Gene Cloning• Techniques for gene cloning enable scientists
to prepare multiple identical copies of gene-sized pieces of DNA.
• Most methods for cloning pieces of DNA share certain general features.– For example, a foreign gene is inserted into a
bacterial plasmid and this recombinant DNA molecule is returned to a bacterial cell.
– Every time this cell reproduces, the recombinant plasmid is replicated as well and passed on to its descendents.
– Under suitable conditions, the bacterial clone will make the protein encoded by the foreign gene.
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• One goal may be to produce a protein product for use.
• A second goal may be to prepare many copies of the gene itself.– This may enable scientists to determine the
gene’s nucleotide sequence or provide an organism with a new metabolic capability by transferring a gene from another organism.
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Restriction Enzymes
• In nature, bacteria use restriction enzymes to cut foreign DNA, such as from phages or other bacteria.
• Most restrictions enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific point in these sequences.
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• Restriction enzyme cleaves DNA at specific sequence of bases called a restriction site.– often a symmetrical series of four to eight bases
on both strands running in opposite directions.– restriction site on one strand is 3’-CTTAAG-5’, the
complementary strand is 5’-GAATTC-3
• Restriction enzymes cut the sugar phosphate backbone bonds of both DNA strands, creating single-stranded ends, sticky ends.– These extensions will form hydrogen-bonded
base pairs with complementary single-stranded stretches on other DNA molecules cut with the same restriction enzyme
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• Recombinant plasmids--produced by splicing restriction fragments from foreign DNA into plasmids.– Plasmid is a circular piece of DNA found in
bacteria and contain genes.– Plasmids can be used to insert DNA from
another organism into a bacterial cell.
• Then, as a bacterium carrying a recombinant plasmid reproduces, the plasmid replicates within it.
Recombinant DNA vectors
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
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• Cloning a gene into a bacterial plasmid can be divided into five steps.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Fig. 20.3
Blue colonies White colonies
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• When the source of DNA is small or impure, the polymerase chain reaction (PCR) is quicker and more selective. (limitation of PCR -- produces short DNA segments within a gene and not entire genes.)
• This technique can quickly amplify any piece of DNA without using cells.
• Devised in 1985, PCR has had a major impact on biological research and technology.
The polymerase chain reaction (PCR) clones DNA entirely in vitro
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
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• The DNA is incubated in atest tube with special DNA polymerase, a supply of nucleotides,and short pieces ofsingle-stranded DNA as a primer.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Fig. 20.7
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PCR
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• PCR can make billions of copies of a targeted DNA segment in a few hours.– This is faster than cloning via recombinant
bacteria.
• PCR, a three-step cycle: – heating, – cooling, – replication, – brings about a chain reaction that produces an
exponential number of DNA molecules.
• PCR is so specific and powerful that only minute amounts of DNA need be present in the starting material
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• PCR has amplified DNA from a variety of sources:– fragments of ancient DNA from a 40,000-year-
old frozen wooly mammoth,– DNA from tiny amount of blood or semen found
at the scenes of violent crimes,– DNA from single embryonic cells for rapid
prenatal diagnosis of genetic disorders,– DNA of viral genes from cells infected with
difficult-to-detect viruses such as HIV.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
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Human chromosome disorders • High frequency in humans
– most embryos are spontaneously aborted– alterations are too disastrous– developmental problems result from biochemical
imbalance• imbalance in regulatory molecules?
– hormones?
– transcription factors?
• Certain conditions are tolerated– upset the balance less = survivable– characteristic set of symptoms = syndrome
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Chromosomal abnormalities• Incorrect numbers of
chromosomes(Numerical)– Nondisjunction
• chromosomes don’t separate properly during meiosis
• Structural abnormalities– Chromosome mutations
• Deletion
• Inversion
• Duplication
• Translocation
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Numerical Abnormalities:Nondisjunction
• Problems with meiotic spindle cause errors in daughter cells– homologous chromosomes do not separate
properly during Meiosis 1– sister chromatids fail to separate during Meiosis 2– too many or too few chromosomes
2n n
n
n-1
n+1
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Alteration of chromosome number
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Nondisjunction • Baby has wrong chromosome number
– Trisomy • cells have 3 copies of a chromosome
– Monosomy• cells have only 1 copy of a chromosome
n+1 n n-1 n
monosomy2n-1
trisomy2n+1
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Down syndrome• Trisomy 21
– 3 copies of chromosome 21– 1 in 700 children born in U.S.
• Chromosome 21 is the smallest human chromosome– but still severe effects
• Frequency of Down syndrome correlates with the age of the mother
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Down syndrome & age of mother
Mother’s age
Incidence of Down Syndrome
Under 30 <1 in 1000
30 1 in 900
35 1 in 400
36 1 in 300
37 1 in 230
38 1 in 180
39 1 in 135
40 1 in 105
42 1 in 60
44 1 in 35
46 1 in 20
48 1 in 16
49 1 in 12
Rate of miscarriage due to amniocentesis: 1970s data
0.5%, or 1 in 200 pregnancies
2006 data<0.1%, or 1 in 1600 pregnancies
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Sex chromosomes abnormalities
• Human development more tolerant of wrong numbers in sex chromosome
• But produces a variety of distinct syndromes in humans– XXY = Klinefelter’s syndrome male – XXX = Trisomy X female– XYY = Jacob’s syndrome male– XO = Turner syndrome female
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• 2X and 1Y– one in every 2000 live births– have male sex organs, but are
sterile– feminine characteristics
• some breast development• lack of facial hair
– tall– normal intelligence
Klinefelter’s syndrome
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Klinefelter’s syndrome
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Jacob’s syndrome male• 1X and 2 Y
– 1 in 1000 live male births
– extra Y chromosome– slightly taller than
average– more active– normal intelligence, slight learning disabilities– delayed emotional immaturity– normal sexual development
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Trisomy X• 3 X
– 1 in every 2000 live births– produces healthy females
• Why?• all but one X chromosome is inactivated
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Turner syndrome• 1X
– 1 in every 5000 births– varied degree of effects – webbed neck– short stature– sterile
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Changes in chromosome structure
• Deletion– loss of a chromosomal segment
• Duplication– repeat a segment
• Inversion– reverses a segment
• Translocation– move segment from one chromosome to
another
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sin
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46,XY,t(8;9)(q24.3;q22.1)
Translocation
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FISH analysis: abl/bcr Genes on Diploid Cells and Ph Positive CML Cells
Normal
Translocation
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Genetics Laboratory• Cytogenetics
Tissue culture
Harvesting/Slide Preparation
KaryotypingResults / Interpretation
Report
FISH
Analysis
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Genetic testing• Amniocentesis in 2nd trimester
– sample of embryo cells– stain & photograph chromosomes
• Analysis of karyotype
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Karyotyping
Karyotype of a normal male
Chromosome Spread
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Dosage CompensationDo males have half as much of the products of
genes on the X as do females? NO!!
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X Inactivation
Interphase: Chromomes can’t be stained, but a dark-staining body is visible in the nuclei of
cells of female mammals
Barr Body: Inactive X
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Which X gets inactivated?Mary Lyon & Lianne Russell
(1961) proposed that one or other of X becomes inactivated at a particular time in early development. Within each cell,which X becomes inactivated is random.
As development proceeds, all cells arising by cell division after that time have same X inactivated.
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In 64-cell embryos
Adult female mammals have two copies of each gene on the X chromosome.