Transcript Library types

Other forms of cloning and
analysis
• PCR
• Restriction mapping
– The human genome project
• In situ methodologies
– These adapt southern, northern and western
techniques to assess genes and gene expression in
cells directly
• Sister Chromatid Exchange
• An example from start to finish
– Cloning the gene responsible for alkaptonuria
What PCR can do
• It can specifically amplify any DNA sequence
from out of a background of contaminating
sequence
• The only specific reagents necessary are primers
that define the amplified region
– The PCR product will geometrically increase in
quantity as a sequence between these two primers
• PCR is conceptually a method of chemically
cloning a DNA sequence
PCR advantages over cloning
• Faster
– Days to weeks to rescue a DNA sequence using
standard techniques
– PCR takes a day from design to completion
• The actual reaction takes a few hours
• Cheaper
– In time and materials
• More versatile
– The polymerase chain reaction can be adapted
to fuse proteins, and create deletions, insertions
and substitution mutations with the same
economy and speed as using PCR for cloning
purposes
PCR disadvantages
• Errors
– This used to be more of a problem than it currently is,
however the thermostable polymerase Taq has no
editing function, and therefore cannot correct errors of
incorporation
– In addition, the reaction is carried out at 72C and is
raised to 98C or higher. This increases oxidative
damage to the DNA
• Size
– PCR products rarely get over 10 kb, and are usually
only a few thousand bases long
• PCR products are difficult to clone
– PCR products are often used as inserts for plasmid
clones because of their ease of preparation, BUT
• Taq polymerase adds an A onto the 3’ end of its product
Restriction
mapping
• The strategy may be employed to
map an entire genome
• Clones contain multiple
restriction sites
• The arrangement of the restriction
sites may be determined by
cutting DNA with single and
multiple enzymes followed by
size assessment on an agarose gel
– Enzyme 1 cuts the linear DNA twice
and enzyme 2 cuts once
– The relative arrangement of the
restriction sites is logically given by
the change in sizes of the fragments
following a double digest
– 3 kb becomes 1+2
– 10 kb becomes 8+2
– 7 kb becomes 6+1
The human genome project
• This was an intensive effort to clone and sequence the entire
human genome
– Once sequenced the data was to be analyzed and turned over to the
public
– It was carried out by a national consortium of federal and
University laboratories
• It became a competition with a private company: Celera
– The “race” was essentially a dead heat, with the private company
claiming patent rights over the sequences they obtained first and
the consortium turning its data over to the public domain
• With everything cloned and sequenced, it is now possible to “clone by
phone” any gene of piece of human DNA if some sequence data is acquired
or if the chromosomal location of the gene is known
The approaches to cloning the
human genome
Human genomic libraries
were made and either
– Systematically
organized and
assembled into a map
– "Shotgun" sequenced
The NIH approach
• Laboratories were assigned
specific sequences such as
chromosomes to work on
– Chromosome specific
libraries were generated
• The library was mapped
using restriction enzymes
– Each clone within the library
was oriented with relation to
the other clones
• This created an entire map of
the chromosome
– The clones were sequenced
The
Celera
approach
• A human genomic library was used as a
source of clones for sequencing
purposes and the clones sequenced
without regard for a map
• The overall sequence of the genome was
assembled because of overlap between
the clones
• The general location of a gene can be determined in humans often
using standard genetics
– We will talk about genetic mapping later
• To determine the exact location of a gene on a chromosome, cloned
DNA may be hybridized to a metaphase chromosome squash
directly
– The DNA probes are fluorescence labeled and are very large (50 kb)
• In-situ hybridization both locates the gene and can assess problems
with the gene
– Below Chromosome 7 is missing DNA near the centromere in Williams
syndrome
In situ
hybridization
techniques
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Sister chromatid
exchange (SCE)
• Homologous recombination
takes place during mitosis as
well as meiosis
– But homologous chromosomes
do not line up
– Instead the sister chromatids
exchange arms
– Since the arms are identical it
has in theory no genetic effect
– However SCE increases under
conditions of increased
mutagenesis
• Exposure to mutagens
• Predisposing genetic conditions
– Since it can be detected it serves
as a marker for those conditions
Detecting SCE
• In culture cells are grown in
the presence of
bromodeoxyuridine (the
thymidine analog) for two
rounds of cell division
• Cells are then used to make
metaphase chromosomes
• One round of replication
produces two sister
chromatids that contain half
BUdR and half thymidine
• The second round of
replication produces one sister
chromatid that contain half
BUdR and half thymidine,
and another sister chromatid
that contains only BUdR.
Normal
Detecting SCE II
• Cells after the second
round of division will
therefore have only one of
the four DNA strands of a
metaphase chromosome
labeled with thymidine
High frequency SCE
– Metaphase spreads are made
and stained with a dye that
stains thymidine containing
strands more than BudR
containing strands
– SCE is visible by the
exchange of arms in the
metaphase chromosomes
Genbank
• This is a national computerized database containing
sequence from any source including
– mRNA
– Genomic clones
– Engineered DNA (often patented)
• It includes relevant data as to who has the clone (so you can
get some – it’s free!)
• If you have a sequence, you can search Genbank and
determine if your sequence matches anything that is known
• With the completion of the human genome project, all
known human sequence is in the database
– The sequence information permits the design of PCR primers and
therefore amplification of any sequence in the human genome
Homogentisic acid 1,2 dioxygenase
(Or HGO to the cognoscenti)
• In 1902 Archibald Garrod
described the disease
alkaptonuria as a buildup of
homogentisic acid
– Alkaptonuria is first recognized
when urine turns black on standing
– There’s Archie
– The pathway for breakdown of
homogentisic acid was
determined, and 10 years later
Garrod also proposed that the
defect was in the enzyme that
converted homogentisic acid to
maleylaectoacetic acid
• That would be HGO
• He was right
Fast forward 84 years to 1992
• Using genetic techniques, the gene for HGO
was mapped in humans to chromosome 3
– Specifically 3q2, or the second band on the
long arm of the chromosome
• 1995, a gene for HGO was cloned from the
yeast
– The sequence was used to search genbank and a
human clone was discovered that was 52%
similar to the yeast gene
Confirmation of a
successful clone-byphone
• The cataloged candidate HGO cDNA
clone was recloned into an expression
vector
– Knowing the sequence permitted alignment of
the open reading frame with the ShineDelgarno sequence of the expression vector
– This candidate HGO clone made protein in
bacteria with HGO activity
• A northern blot using the cloned DNA as
probe and liver RNA as target resulted in
a single band reflecting the HGO mRNA
– HGO is a liver enzyme
– These data indicate the DNA represents the
HGO gene
Further studies
• The cDNA was used as probe to rescue
a genomic clone
• In situ hybridization using the clone
paints chromosome 3 at band q2
• DNA taken from people with
alkaptonuria was amplified using HGO
specific primers and PCR
– In a few hours, enough DNA was available
for sequencing
– HGO mutations responsible were
identified and could be tracked in families
• The probability of transmission can be
determined in couples that are having
kids
– The genetic constitution of a fetus in utero
can also be addressed
• Alkaptonuria is not a lethal defect
An ethical
dilemma
– Arthritis due to the buildup of black pigment
in the joints
– It sometimes makes its appearance late in life
– It is treatable with high doses of vitamin C
• This decreases the affinity of the metabolite
for cartilage and may affect the arthritis
– And other drugs
• But if you knew early in term that a fetus
was going to have no functioning HGO,
what would you do?
• As well, if you could stop the birth of the
four sisters with androgen insensitivity?
• Science gives you, the physician, the
power of prediction, but does not tell
you how or when to use it