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Jelly fish “GFP”
Transformed vertebrates
DNA Manipulation, Gene
Cloning and Karyotyping
Gene Cloning
• Techniques for gene cloning enable scientists
to prepare multiple identical copies of genesized 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.
• 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.
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.
• 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
Recombinant DNA vectors
• 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.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Cloning a gene
into a bacterial
plasmid can be
divided into five
steps.
Blue colonies
White colonies
Fig. 20.3
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
The polymerase chain reaction
(PCR) clones DNA entirely in vitro
• 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.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• The DNA is
incubated in a
test tube with
special DNA
polymerase, a
supply of
nucleotides,
and short
pieces of
singlestranded DNA
as a primer.
Fig. 20.7
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
PCR
• 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
• PCR has amplified DNA from a variety of
sources:
– fragments of ancient DNA from a 40,000-yearold 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
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
Chromosomal abnormalities
• Incorrect numbers of
chromosomes(Numerical)
– Nondisjunction
• chromosomes don’t separate properly
during meiosis
• Structural abnormalities
– Chromosome mutations
• Deletion
• Inversion
• Duplication
• Translocation
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-1
n
n+1
n
Alteration of chromosome number
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-1
n
n
trisomy
monosomy
2n+1
2n-1
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
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
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
Klinefelter’s syndrome
• 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
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
Trisomy X
• 3X
– 1 in every 2000 live births
– produces healthy females
• Why?
• all but one X chromosome is inactivated
Turner syndrome
• 1X
– 1 in every 5000 births
– varied degree of effects
– webbed neck
– short stature
– sterile
replication
crossing over
error of
error of
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
Translocation
46,XY,t(8;9)(q24.3;q22.1)
FISH analysis: abl/bcr Genes on
Diploid Cells and Ph Positive
Translocation
CML Cells
Normal
Genetics Laboratory
• Cytogenetics
Tissue culture
Harvesting/Slide Preparation
FISH
Analysis
Karyotyping
Results /
Interpretation
Report
Genetic testing
• Amniocentesis in 2nd trimester
– sample of embryo cells
– stain & photograph chromosomes
• Analysis of karyotype
Karyotyping
Chromosome Spread
Karyotype of a normal
male
Dosage Compensation
Do males have half as much of the products of
genes on the X as do females?
NO!!
X Inactivation
Barr Body: Inactive X
Interphase: Chromomes can’t be stained, but a
dark-staining body is visible in the nuclei of
cells of female mammals
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.
In 64-cell embryos
Adult female
mammals have
two copies of
each gene on
the X
chromosome.