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Worksheetsbiochem of nucleic acids
Total questions: 208
Worksheet time: 7hrs 56mins
Griffith
repeated Griffith’s experiment but in test tubes
subjected it to digestion with various enzymes
found the transforming substance was DNA
injected smooth (S strain) and rough (R strain) in mice
found the heat-killed bacteria was probably the gene virulence itself
missing piece was the chemical nature of the transforming substance
wanted to know if genes reside in protein or DNA
DNA is rich in phosphorus
Protein has no phosphorus
labeled
DNA of the phage (P32)
Proteins of the phage (S35)
the genes of the phage are made of DNA
DNA is composed of 2 antiparallel strands
DNA strand contains info for the synthesis of the other strand
DNA replication is semiconservative
used 15N (heavy N) and 14N (lighter N)
DNA replication results in new DNA duplex molecules in which 1 strand is from the parent duplex and the other is completely new
Avery, Macleod, McCarty
repeated Griffith’s experiment but in test tubes
subjected it to digestion with various enzymes
found the transforming substance was DNA
total amount of purines (A+G) is = to total amount of pyrimidines (T+C)
purines has a double ring
pyrimidines are single rings
Determined the molecular weight of DNA
used Autoradiography
used a radioactive label and incorporated it into a substance
made E.coli radioactive
wanted to know if genes reside in protein or DNA
DNA is rich in phosphorus
Protein has no phosphorus
labeled
DNA of the phage (P32)
Proteins of the phage (S35)
the genes of the phage are made of DNA
Hershey-Chase
DNA is composed of 2 antiparallel strands
DNA strand contains info for the synthesis of the other strand
DNA replication is semiconservative
used 15N (heavy N) and 14N (lighter N)
DNA replication results in new DNA duplex molecules in which 1 strand is from the parent duplex and the other is completely new
identified SV40
origin of replication
DNA replication proceeded bidirectional
used EcoRI (Restriction enzyme) to cleave replicating SV40 DNA molecules at a unique
repeated Griffith’s experiment but in test tubes
subjected it to digestion with various enzymes
found the transforming substance was DNA
wanted to know if genes reside in protein or DNA
DNA is rich in phosphorus
Protein has no phosphorus
labeled
DNA of the phage (P32)
Proteins of the phage (S35)
the genes of the phage are made of DNA
Chargaff
repeated Griffith’s experiment but in test tubes
subjected it to digestion with various enzymes
found the transforming substance was DNA
found all necessary ingredients required for the synthesis of E.coli DNA in vitro
A=T
G=C
total amount of purines (A+G) is = to total amount of pyrimidines (T+C)
purines has a double ring
pyrimidines are single rings
injected smooth (S strain) and rough (R strain) in mice
found the heat-killed bacteria was probably the gene virulence itself
missing piece was the chemical nature of the transforming substance
DNA
phosphate
attached to the 5’ carbon of the sugar by a phosphodiester linkage
no hydroxyl group at the 2’ position
2-deoxyribose in DNA
a purine or pyrimidine base attached to the 1’ carbon atom of the sugar by an N- glycosidic bond
humidity is high
DNA is in B form
humidity is low
DNA is in A form
B and A are right handed helices
Z DNA is a left handed helix
helix destabilizing proteins
bind only to separated single stranded DNA
prevent from base pairing back together
a “sealing” enzyme
nucleotide
nucleoside+phosphate
sugar linked to a base
no phosphate
nucleoside
sugar linked to a base, no phosphate
sugar lined to a base
no phosphate
what pair is more stable
G-C
A-T
A-U
DNA Watson and Crick
DNA molecule consist of 2 polynucleotide chains wound around each other in a right handed double helix
true
false
DNA Watson and Crick
the 2 chains are parallel, both go in the 5' to 3' direction
true
false
DNA watson and crick
sugar phosphate backbones are on the outside of the double helix
bases are oriented towards the central axis
true
false
watson and crick
the bases of both chains are round structures oriented perpendicular to the long axis of the DNA
true
false
WATSON AND CRICK
the bases opposite strand are bonded together by relatively weak hydrogen bonds
weak hydrogen bonds make it easier to separate the 2 strands
true
false
watson and crick
the 2 sugar phosphate backbones of the double helix are not equally spaced along the helical axis
results in groves of unequal sizes between the backbones
called major and minor grooves
true
false
DNA A
humidity is high, right handed, minor and major
humidity is low, left handed, minor
humidity is low, right handed, major and minor
humidity is low, left handed, major and minor
DNA Z
left handed, minor
right handed, minor
left handed, major and minor
right handed, major and minor
DNA B
humidity is low, left handed, major and minor
humidity is high, left handed, major and minor
humidity is high, right handed, major and minor
humidity is low, right handed, major and minor
Meselson and Stahl
DNA is composed of 2 antiparallel strands
DNA strand contains info for the synthesis of the other strand
DNA replication is semiconservative
used 15N (heavy N) and 14N (lighter N)
DNA replication results in new DNA duplex molecules in which 1 strand is from the parent duplex and the other is completely new
Determined the molecular weight of DNA
used Autoradiography
used a radioactive label and incorporated it into a substance
made E.coli radioactive
revealed that the intact chromosomes of E.coli is a single high circle
radioactive DNA isolated from cells during replication showed an extra loop
the structures contained eyes (bubbles)
resembling theta
both strands are replicated at the same time
1 or both ends of the loop are dynamic points
repeated Griffith’s experiment but in test tubes
subjected it to digestion with various enzymes
found the transforming substance was DNA
wanted to know if genes reside in protein or DNA
DNA is rich in phosphorus
Protein has no phosphorus
labeled
DNA of the phage (P32)
Proteins of the phage (S35)
the genes of the phage are made of DNA
DNA replication
results in new DNA duplex molecules in which 1 strand is from the parent duplex and the other is completely new
true
false
Cairns
Determined the molecular weight of DNA
used Autoradiography
used a radioactive label and incorporated it into a substance
made E.coli radioactive
revealed that the intact chromosomes of E.coli is a single high circle
radioactive DNA isolated from cells during replication showed an extra loop
the structures contained eyes (bubbles)
resembling theta
both strands are replicated at the same time
1 or both ends of the loop are dynamic points
DNA is composed of 2 antiparallel strands
DNA strand contains info for the synthesis of the other strand
DNA replication is semiconservative
used 15N (heavy N) and 14N (lighter N)
DNA replication results in new DNA duplex molecules in which 1 strand is from the parent duplex and the other is completely new
wanted to know if genes reside in protein or DNA
DNA is rich in phosphorus
Protein has no phosphorus
labeled
DNA of the phage (P32)
Proteins of the phage (S35)
the genes of the phage are made of DNA
njected smooth (S strain) and rough (R strain) in mice
found the heat-killed bacteria was probably the gene virulence itself
missing piece was the chemical nature of the transforming substance
replication forks
where parental DNA is being unwound and the separated strands are quickly replicated
true
false
replication of bacterial chromosomes is bidirectional or only one direction
one direction
bidirectional
Salzman
DNA is composed of 2 antiparallel strands
DNA strand contains info for the synthesis of the other strand
DNA replication is semiconservative
used 15N (heavy N) and 14N (lighter N)
DNA replication results in new DNA duplex molecules in which 1 strand is from the parent duplex and the other is completely new
total amount of purines (A+G) is = to total amount of pyrimidines (T+C)
purines has a double ring
pyrimidines are single rings
identified SV40
origin of replication
DNA replication proceeded bidirectional
used EcoRI (Restriction enzyme) to cleave replicating SV40 DNA molecules at a unique site
SV40 DNA contains only 1 single EcoRI site
only one single replicating bubble, indicated a single origin of replication
bubble grew at both ends
both replicating forks were moving away from the single origin
DNA molecules can coil and bend in space, leading to changes in topology
formation of supercoils
true
false
Topisomerases
class of enzyme that control DNA topology
perform essential functions at several different steps in replication of DNA
true
false
Type 1 topoisomerase
changes the degree of supercoiling of DNA by causing single strand breaks and re-ligation
bacterial gyros
cause double strand breaks
implicated in recombination events
impacted in process of segregation of the newly replicated chromosomes
Type 2 topisomerases
implicated in recombination events
impacted in process of segregation of the newly replicated chromosomes
bacterial gyros
cause double strand breaks
changes the degree of supercoiling of DNA by causing single strand breaks and re-ligation
Topisomerase 3
impacted in process of segregation of the newly replicated chromosomes
changes the degree of supercoiling of DNA by causing single strand breaks and re-ligation
bacterial gyros
cause double strand breaks
implicated in recombination events
Topiomerase 4
bacterial gyros
cause double strand breaks
impacted in process of segregation of the newly replicated chromosomes
changes the degree of supercoiling of DNA by causing single strand breaks and re-ligation
implicated in recombination events
DNA polymerase 1
repair of damaged DNA
Exonuclease 5’-4’ is missing
de novo synthesis of new stands of DNA
smallest molecules/cell
involved in
repair of damaged DNA
subsidiary role in semiconservative replication
largest molecules/cell
DNA polymerase 2
repair of damaged DNA
Exonuclease 5’-4’ is missing
involved in
repair of damaged DNA
subsidiary role in semiconservative replication
largest molecules/cell
de novo synthesis of new stands of DNA
smallest molecules/cell
DNA polymerase 3
involved in
repair of damaged DNA
subsidiary role in semiconservative replication
largest molecules/cell
de novo synthesis of new stands of DNA
smallest molecules/cell
repair of damaged DNA
Exonuclease 5’-4’ is missing
exonuclease activity
5’-3’ exonuclease servers a proofreading function
removes incorrectly matched bases, so polymerase can try again
true
false
exonuclease activity
5’ exonuclease activity accomplishes “nick translation”
TRUE
FALSE
All prokaryotic and eukaryotic DNA polymerases share the same fundamental type of synthetic activity
TRUE
FALSE
Kornberg
found all necessary ingredients required for the synthesis of E.coli DNA in vitro
their DNA is biological inactive
requires a primer to provide a free 3’-OH end that can be extended by addition of nucleotides
identified SV40
origin of replication
DNA replication proceeded bidirectional
used EcoRI (Restriction enzyme) to cleave replicating SV40 DNA molecules at a unique site
SV40 DNA contains only 1 single EcoRI site
only one single replicating bubble, indicated a single origin of replication
bubble grew at both ends
Determined the molecular weight of DNA
used Autoradiography
used a radioactive label and incorporated it into a substance
made E.coli radioactive
revealed that the intact chromosomes of E.coli is a single high circle
radioactive DNA isolated from cells during replication showed an extra loop
the structures contained eyes (bubbles)
resembling theta
both strands are replicated at the same time
1 or both ends of the loop are dynamic points
DNA is composed of 2 antiparallel strands
DNA strand contains info for the synthesis of the other strand
DNA replication is semiconservative
used 15N (heavy N) and 14N (lighter N)
DNA replication results in new DNA duplex molecules in which 1 strand is from the parent duplex and the other is completely new
PRIMER
is a short sequence that is paired with one strand of DNA and provides a free 3’-OH end at which a DNA polymerase starts synthesis of deoxyribonucleotide chain
TRUE
FALSE
DNA helicases
enzymes bind to a single stranded DNA near the replication fork and then move into the neighbouring double stranded region, forcing the strands apart in effect, unwinding the double helix
requires energy (ATP)
make single stranded cut
one the strand separated allow single stranded DNA binding (SSB) proteins bind, preventing reformation of the double helix
enzymes bind to a single stranded DNA near the replication fork and then move into the neighbouring double stranded region, forcing the strands apart in effect, unwinding the double helix
make single stranded cut
one the strand separated allow single stranded DNA binding (SSB) proteins bind, preventing reformation of the double helix
enzymes bind to a single stranded DNA near the replication fork and then move into the neighbouring double stranded region, forcing the strands apart in effect, unwinding the double helix
requires energy (ATP)
one the strand separated allow single stranded DNA binding (SSB) proteins bind, preventing reformation of the double helix
enzymes bind to a single stranded DNA near the replication fork and then move into the neighbouring double stranded region, forcing the strands apart in effect, unwinding the double helix
requires energy (ATP)
make single stranded cut
SSB proteins
do not precent the base from pairing back together
bind to double stranded DNA
helix destabilizing proteins
bind only to separated single stranded DNA
prevent from base pairing back together
bind to single stranded DNA
Dna protein
bind to specific nucleotide sequences at the origin of replication (rich in AT base pairs)
ATP requiring process causes the double stranded DNA to melt
the strand separate, forming localized regions of single stranded DNA
requires ATP
requires no ATP
RNA primase
not in DNA polymerase 3
makes very short piece of RNA by base pairing RNA nucleotides with template DNA
carried out by RNA dependent RNA primase
DNA ligase
makes very short piece of RNA by base pairing RNA nucleotides with template DNA
ATP requiring process causes the double stranded DNA to melt
the strand separate, forming localized regions of single stranded DNA
a “sealing” enzyme
join any gaps where adjacent nucleotides on one strand have not been covalently joined
Need energy (ATP or phosphate bond of NAD+)
Discontinuous DNA replication
each fragment grows in the 5'-3' direction
each fragment grows in the 3'-5' direction
Discontinuous DNA replication
leading strand
is synthesized discontinuously in the form of short fragments
is synthesized continuously as the parental duplex is unwound
Discontinuous DNA replication
lagging strand
is synthesized discontinuously in the form of short fragments
is synthesized continuously as the parental duplex is unwound
Fidelity of DNA replication
prokaryotes
DNA replication beings at a single, fixed location in this molecule, the replication origin
done in no more than 40 minutes
process takes a month
many replication origins
replication begins at some origins earlier in S phase than at others, but the process is completed by the end of S phase
Fidelity of DNA replication
eukaryotes
process takes a month
many replication origins
replication begins at some origins earlier in S phase than at others, but the process is completed by the end of S phase
when replication nears completion, bubbles form and fuse, forming 2 new molecules
DNA replication beings at a single, fixed location in this molecule, the replication origin
done in no more than 40 minutes
process takes a month
many replication origins
replication begins at some origins earlier in R phase than at others, but the process is completed by the end of R phase
when replication nears completion, bubbles form and fuse, forming 2 new molecules
process is very quick
many replication origins
replication begins at some origins earlier in S phase than at others, but the process is completed by the end of S phase
when replication nears completion, bubbles form and fuse, forming 2 new molecules
DNA mutations
can occur spontaneously
true
false
how many types of point mutations
2
3
5
4
Transitions
involves the change of
a purine to a pyrimidine
a pyrimidine to a purine
addition of a new base pair
involve the change of
1 purine to another
1 pyrimidine to another
removal of a new base pair
Transversions
involve the change of
1 purine to another
1 pyrimidine to another
addition of a base pair
involves the change of
a purine to a pyrimidine
a pyrimidine to a purine
removal of a base pair
Deletions and insertions are mutations involving either the removal or the addition of a base pair
lead to changes in the reading frame, they are frameshift mutations
true
false
Physical agents that bring about mutations in DNA include
X-rays
leads to formation of pyrimidine dimers
gives a new entity
causes nicks or breaks in a single strand
Physical agents that bring about mutations in DNA include
UV radiation
leads to formation of pyrimidine dimers
Thymine dimers form when 2 consecutive pyrimidines in one strand become covalently linked to each other
causes nicks or breaks in a single strand
gives a new entity
Many mutagens are also carcinogens
true
false
Chemical mutagenesis may result from modification of bases due to
oxidative deamination of nitrous acid or precursors of nitrous acid
gives a new entity
reactive compounds that can transfer methyl or ethyl groups to a DNA bade
cross linking of DNA strands
alteration of the helix by intercalating agents
planar molecules that become inserted between adjacent base pairs and lead to insertion or deletion of one or more base pairs
leads to formation of pyrimidine dimers
Chemical mutagenesis may result from modification of bases due to alkylation by alkylating agents
gives a new entity
causes nicks or breaks in a single strand
reactive compounds that can transfer methyl or ethyl groups to a DNA bade
cross linking of DNA strands
alteration of the helix by intercalating agents
planar molecules that become inserted between adjacent base pairs and lead to insertion or deletion of one or more base pairs
Direct reversal of damaging reaction
most damage to DNA is repaired by removal of the damaged bases followed by resynthesis of the excised region
some lesions in DNA can be repaired by direct reversal of the damage
more efficient way of dealing with specific type of DNA damage
only a few types can be repaired this way
pyrimidine dimers resulting from exposure to UV
alkylated guanine residues that have been modified by the addition of methyl or ethyl groups at the O^6 position of the purine ring
repair of pyrimidine dimers by Photoreavtivation
is a bit universal
many species including humans lack this mechanism of DNA repair
Repair by O^6 meghylguanine methyltransferase
most common form of DNA repair
complex system
employs some of the enzymes of DNA replication
many different types of damage are repaired including pyrimidine dimers
involves removing a damaged DNA segment and replacing it with the normal base sequence
first
DNA repair endonuclease or endnuclease-containg enzyme complex
recognizes
binds to
excises the damages base or bases in DNA
second
DNA polymerase fills in the gap by using the undamaged complementary strand of DNA as a template
third
DNA ligase seals the break left by DNA polymerase to complete the repair process
corrects errors introduced during DNA replication by identifying mismatched nucleotides
mechanism is based on the occurrence of methylated bases of DNA
during replication, parental DNA is fully methylated
daughter DNA is under methylated for a brief period of time
due to DNA methylation lags behind DNA synthesis
capacity to recognize
unmethylated sequences
mismatched base pairs in the newly synthesized daughter strand
repair involves excising a segment of the daughter strand and includes the mismatched
excised segment is replaced by a new segment contains the correct base
Excision repair
most common form of DNA repair
complex system
employs some of the enzymes of DNA replication
many different types of damage are repaired including pyrimidine dimers
involves removing a damaged DNA segment and replacing it with the normal base sequence
first
DNA repair endonuclease or endnuclease-containg enzyme complex
recognizes
binds to
excises the damages base or bases in DNA
second
DNA polymerase fills in the gap by using the undamaged complementary strand of DNA as a template
third
DNA ligase seals the break left by DNA polymerase to complete the repair process
a complex set of cellular changes
induced in bacteria subjected to lethal mutagens
UV
alkylating agents
cells cease to divide and increase their capacity to repair damaged DNA
the whole battery of DNA repair, recombination and replication proteins are synthesized
is a risky attempt to escape lethal effects of heavily damaged DNA
damaged DNA undergo replication though all the damage has not been repaired
the damaged region is skipped during replication
the gap is filled by DNA polymerase and ligase
2 major types of excision repair
base excision
systems remove larger defects like thymine dimers
systems remove abnormal or chemically modified bases from DNA
2 major types of excision repair
nucleotide excision repair
systems remove abnormal or chemically modified bases from DNA
systems remove larger defects like thymine dimers
Excinuclease activity in E.coli require the products of 3 genes (UvrA, UvrB, UvrC)
true
false
nucleotide excision repair involves about _____ times as Many proteins
3
2
4
Xeroderma pigmentosum (XP)
no treatment
inherited, associated with defective nucleotide excision repair
hypersensitive to UV light
exhibit stunted growth
autosomal recessive disease
UV induced pyrimidine dimers and chemical adducts not excised from DNA
DNA ligase and repair enzymes are defective
develop aplastic anemia
pigmentary changes in skin
autosomal recessive disease
lack of balance and slurred speech
muscle coordination defect
high frequency of chromosome breaks leading to translocations and inversions
sensitive to sun and UV light
a rare hereditary disease in humans
due to a deficiency of the endonuclease that introduce nicks into damaged sections of DNA
Cockayne syndrome (CS)
no treatment
inherited, associated with defective nucleotide excision repair
hypersensitive to UV light
exhibit stunted growth
sensitive to sun and UV light
a rare hereditary disease in humans
due to a deficiency of the endonuclease that introduce nicks into damaged
autosomal recessive disease
lack of balance and slurred speech
muscle coordination defect
high frequency of chromosome breaks leading to translocations and inversions
autosomal recessive disease
involves deficiency in DNA ligase
pre- and postnatal growth deficiency
sun-sensitive skin
chromosome instability
diabetes mellitus
immunodeficiency
ataxia telangiectasia (AT)
autosomal recessive disease
lack of balance and slurred speech
muscle coordination defect
high frequency of chromosome breaks leading to translocations and inversions
autosomal recessive disease
involves deficiency in DNA ligase
pre- and postnatal growth deficiency
sun-sensitive skin
chromosome instability
diabetes mellitus
immunodeficiency
autosomal recessive disease
UV induced pyrimidine dimers and chemical adducts not excised from DNA
DNA ligase and repair enzymes are defective
develop aplastic anemia
pigmentary changes in skin
fanconi’s anemia (FA)
autosomal recessive disease
UV induced pyrimidine dimers and chemical adducts not excised from DNA
DNA ligase and repair enzymes are defective
develop aplastic anemia
pigmentary changes in skin
autosomal recessive disease
lack of balance and slurred speech
muscle coordination defect
high frequency of chromosome breaks leading to translocations and inversions
autosomal recessive disease
involves deficiency in DNA ligase
pre- and postnatal growth deficiency
sun-sensitive skin
chromosome instability
diabetes mellitus
immunodeficiency
bloom’s syndrome (BS)
autosomal recessive disease
UV induced pyrimidine dimers and chemical adducts not excised from DNA
DNA ligase and repair enzymes are defective
develop aplastic anemia
pigmentary changes in skin
autosomal recessive disease
lack of balance and slurred speech
muscle coordination defect
high frequency of chromosome breaks leading to translocations and inversions
autosomal recessive disease
involves deficiency in DNA ligase
pre- and postnatal growth deficiency
sun-sensitive skin
chromosome instability
diabetes mellitus
immunodeficiency
Mismatch DNA repair
a complex set of cellular changes
induced in bacteria subjected to lethal mutagens
UV
alkylating agents
cells cease to divide and increase their capacity to repair damaged DNA
the whole battery of DNA repair, recombination and replication proteins are synthesized
is a risky attempt to escape lethal effects of heavily damaged DNA
corrects errors introduced during DNA replication by identifying mismatched nucleotides
mechanism is based on the occurrence of methylated bases of DNA
during replication, parental DNA is fully methylated
daughter DNA is under methylated for a brief period of time
due to DNA methylation lags behind DNA synthesis
capacity to recognize
unmethylated sequences
mismatched base pairs in the newly synthesized daughter strand
repair involves excising a segment of the daughter strand and includes the mismatched
excised segment is replaced by a new segment contains the correct base
damaged DNA undergo replication though all the damage has not been repaired
the damaged region is skipped during replication
the gap is filled by DNA polymerase and ligase
SOS repair
a complex set of cellular changes
induced in bacteria subjected to lethal mutagens
UV
alkylating agents
cells cease to divide and increase their capacity to repair damaged DNA
the whole battery of DNA repair, recombination and replication proteins are synthesized
is a risky attempt to escape lethal effects of heavily damaged DNA
corrects errors introduced during DNA replication by identifying mismatched nucleotides
mechanism is based on the occurrence of methylated bases of DNA
during replication, parental DNA is fully methylated
daughter DNA is under methylated for a brief period of time
due to DNA methylation lags behind DNA synthesis
capacity to recognize
unmethylated sequences
mismatched base pairs in the newly synthesized daughter strand
repair involves excising a segment of the daughter strand and includes the mismatched
excised segment is replaced by a new segment contains the correct base
damaged DNA undergo replication though all the damage has not been repaired
the damaged region is skipped during replication
the gap is filled by DNA polymerase and ligase
Recombination Repair
most damage to DNA is repaired by removal of the damaged bases followed by resynthesis of the excised region
some lesions in DNA can be repaired by direct reversal of the damage
more efficient way of dealing with specific type of DNA damage
only a few types can be repaired this way
pyrimidine dimers resulting from exposure to UV
alkylated guanine residues that have been modified by the addition of methyl or ethyl groups at the O^6 position of the purine ring
repair of pyrimidine dimers by Photoreavtivation
is a bit universal
many species including humans lack this mechanism of DNA repair
Repair by O^6 meghylguanine methyltransferase
a complex set of cellular changes
induced in bacteria subjected to lethal mutagens
UV
alkylating agents
cells cease to divide and increase their capacity to repair damaged DNA
the whole battery of DNA repair, recombination and replication proteins are synthesized
is a risky attempt to escape lethal effects of heavily damaged DNA
damaged DNA undergo replication though all the damage has not been repaired
the damaged region is skipped during replication
the gap is filled by DNA polymerase and ligase
ames test
shows the probability that a substance is a mutagen or carcinogen and the relative mutagenicity or carinogenicty of the substance
histidine requiring (His-) mutants of the bacterium salmonella typhimurim are tested for reverse of His+
100% gurantee
shows the probability that a substance is a mutagen or carcinogen and the relative mutagenicity or carinogenicty of the substance
histidine requiring (His+) mutants of the bacterium salmonella typhimurim are tested for reverse of His-
DNA recombination
makes new combination of genetic info on a new DNA molecule
results in the exchange or transfer of segments of DNA from one chromosome to another or with a chromosome
most are examples of homologous recombination (general recombination)
occurs between any 2 DNA molecules or fragments of the same DNA molecule that have closely related sequences
true
false
in bacteria can occur during
conjugation
DNA fragment becomes incorporated into the chromosome of a recipient bacterial cell (Griffiths experiment)
sexual reproduction
DNA transfer between cells of opposite mating types
Lederberg and Tatum showed that 2 different strains of bacteria with different growth requirements could exchange genes
transfer of DNA segment first becomes incorporated into the phage DNA and from there is transferred to the DNA of the recipient cell
in bacteria can occur during
transformation
transfer of DNA segment first becomes incorporated into the phage DNA and from there is transferred to the DNA of the recipient cell
sexual reproduction
DNA transfer between cells of opposite mating types
Lederberg and Tatum showed that 2 different strains of bacteria with different growth requirements could exchange genes
DNA fragment becomes incorporated into the chromosome of a recipient bacterial cell (Griffiths experiment)
in bacteria can occur during
Transduction
transfer of DNA segment first becomes incorporated into the phage DNA and from there is transferred to the DNA of the recipient cell
DNA fragment becomes incorporated into the chromosome of a recipient bacterial cell (Griffiths experiment)
sexual reproduction
DNA transfer between cells of opposite mating types
Lederberg and Tatum showed that 2 different strains of bacteria with different growth requirements could exchange genes
site specific recombination
rare event
require neither sequence similarity nor the action of any known protein between the recombine DNA’s
only limited sequence
recombine DNA’a and always involves the same DNA regions
short segment of DNA with the remarkable capacity to move from one location in chromosome to another
homologous recombination occurs via crossing over
genetic material is exchanged between homologous chromosomes during meiosis
true
false
illegitamate recombination
only limited sequence
recombine DNA’a and always involves the same DNA regions
rare event
require neither sequence similarity nor the action of any known protein
short segment of DNA with the remarkable capacity to move from one location in chromosome to another
DNA transposition
rare event
require neither sequence similarity nor the action of any known protein between the recombine DNA’s
short segment of DNA with the remarkable capacity to move from one location in chromosome to another
only limited sequence
recombine DNA’a and always involves the same DNA regions
Meselson and Weigle
co-infected E.coli with 2 pages carrying different markers
one grown contain heavy isotopes (15N,13C)
other growth in normal light isotopes (14N,12C)
recombinant viruses were found to have intermediate densities
non-recombinant phages were uniformly light or heavy
recombinant phages contained DNA derived from both parents by breaking and rejoining
DNA is composed of 2 antiparallel strands
DNA strand contains info for the synthesis of the other strand
DNA replication is semiconservative
used 15N (heavy N) and 14N (lighter N)
DNA replication results in new DNA duplex molecules in which 1 strand is from the parent duplex and the other is completely new
wanted to know if genes reside in protein or DNA
DNA is rich in phosphorus
Protein has no phosphorus
labeled
DNA of the phage (P32)
Proteins of the phage (S35)
the genes of the phage are made of DNA
Holiday Model
a model of heterologous recombination
true
false
Holiday Model
STEP 1
1 strand of each double helix breaks
recombination process is recognition and alignment in which the 2 homologous DNA double helices become precisely aligned
pairing process is called synapsis
Each broken strand invaded the opposite double helix and base pairs with the complementary nucleotides of the invaded helix
called invasion
leaves gaps that are sealed by DNA polymerase and DNA ligase
produces Chi (X) structure
Holiday Model
STEP 2
1 strand of each double helix breaks
Each broken strand invaded the opposite double helix and base pairs with the complementary nucleotides of the invaded helix
called invasion
leaves gaps that are sealed by DNA polymerase and DNA ligase
produces Chi (X) structure
recombination process is recognition and alignment in which the 2 homologous DNA double helices become precisely aligned
pairing process is called synapsis
Holiday Model
Step 3
Each broken strand invaded the opposite double helix and base pairs with the complementary nucleotides of the invaded helix
called invasion
leaves gaps that are sealed by DNA polymerase and DNA ligase
produces Chi (X) structure
1 strand of each double helix breaks
2 DNA double helices can rotate
generates different conformations of the holiday structure
4-armed structure for the DNA strand is produced by pulling the 4 chromosome ends apart
Holiday Model
Step 4
2 DNA double helices can rotate
generates different conformations of the holiday structure
4-armed structure for the DNA strand is produced by pulling the 4 chromosome ends apart
holiday intermediate is cut by the enzymes at 2 points in the single stranded DNA region of the branch point
cut can either be vertical or horizontal planes
both occur at equal probability
sealing the nicks leaves 2 DNA duplex with
short stretched of hetroduplex containing 1 strand from each of the recombine partners (no true recombination)
Each broken strand invaded the opposite double helix and base pairs with the complementary nucleotides of the invaded helix
called invasion
leaves gaps that are sealed by DNA polymerase and DNA ligase
produces Chi (X) structure
Holiday Model
Step 5
Each broken strand invaded the opposite double helix and base pairs with the complementary nucleotides of the invaded helix
called invasion
leaves gaps that are sealed by DNA polymerase and DNA ligase
produces Chi (X) structure
holiday intermediate is cut by the enzymes at 2 points in the single stranded DNA region of the branch point
cut can either be vertical or horizontal planes
both occur at equal probability
sealing the nicks leaves 2 DNA duplex with
short stretched of hetroduplex containing 1 strand from each of the recombine partners (no true recombination)
patched duplexes
recombinant DNA
spliced duplexes
2 DNA double helices can rotate
generates different conformations of the holiday structure
4-armed structure for the DNA strand is produced by pulling the 4 chromosome ends apart
Holiday model
explains the events of recombination
does not give any mechanism of exchange reactions
does not give any mechanism of molecular aspects of the process
true
false
RecA : recombinase RecA
drive branch migration and process the holiday structure into recombinant products
binds to single stranded DNA, forms a nucleoprotein filament capable of strain invasion and homologous pairing
binds to the end of a DNA duplex and its helices activity starts to unwind the double helix
RecBCD:
binds to the emerging single strands
drive branch migration and process the holiday structure into recombinant products
enzymatic complex which imitates recombination
RuvA, RuvB, RuvC proteins
binds to single stranded DNA, forms a nucleoprotein filament capable of strain invasion and homologous pairing
drive branch migration and process the holiday structure into recombinant products
binds to the emerging single strands
RecBCD
binds to the end of a DNA duplex and its helices activity starts to unwind the double helix
binds to the emerging single strands
binds to single stranded DNA, forms a nucleoprotein filament capable of strain invasion and homologous pairing
the endonuclease activity of RecBCD
cuts randomly cleaves the single strand DNA
When RecBCD encounters a nucleotide sequence (Chi site)
3’ terminal strand is cleaved just below the 3’ end Chi site
binds to the end of a DNA duplex and its helices activity starts to unwind the double helix
integration of RecBCD with Chi site caused the RecD to become irreversibly altered
true
false
RecBCD no longer expresses nuclease activity towards the 5’ terminal strand
true
false
RecBCD nuclease activity against the 5’ terminal strand increases
TRUE
FALSE
RuvA and B work together as a holiday junction specific helices complex
dissociates the RecA filament
catalyzes branch migration
is an endonuclease that binds at the junction and cuts pairs of DNA strand of similar polarity
splice and patch recombinants
RuvC
dissociates the RecA filament
catalyzes branch migration
is an endonuclease that binds at the junction and cuts pairs of DNA strand of similar polarity
splice and patch recombinants
tetramer fits within the junction point
Recombination
Transposition
involves physical movement of DNA segments from one chromosomal locus to another
occurs in prokaryotes and eukaryotes
range in length from several hundred to tens of thousands of base pairs
can be divided into 3 classes
transposons are autonomous units and each encodes and enzyme called transposase
true
false
transposase catalyzes its own transposition
range in length from several hundred to tens of thousands of base pairs
can be divided into 3 classes
involves physical movement of DNA segments from one chromosomal locus to another
occurs in prokaryotes and eukaryotes
insertion sequence (IS)
promotes changes in DNA (insertion and deletions)
plays vital role in the capacity of bacteria to mutate at a rapid rate
represents the simplest transposable elements
contains a small ds DNA segment that generally contains fewer than 2000 bo
contains a gene that codes for transposase
sometimes a regulatory gene related to the process of transposition, but no other gene
flanked by inverted repeats
identical sequences reading in opposite directions
contain not only the gene for transposase but also a # of other genes not related to transposition
Transposons
larger transposons elements
contain not only the gene for transposase but also a # of other genes not related to transposition
transposes are flanked by inverted repeats (identical sequences, reading in opposite directions)
promotes changes in DNA (insertion and deletions)
plays vital role in the capacity of bacteria to mutate at a rapid rate
accounts for the development of bacterial resistance to antibodies
composite transposons
Transposons and composite transposons
larger transposons elements
contain not only the gene for transposase but also a # of other genes not related to transposition
transposes are flanked by inverted repeats (identical sequences, reading in opposite directions)
are flanked by insertion sequences
transposition
promotes changes in DNA (insertion and deletions)
plays vital role in the capacity of bacteria to mutate at a rapid rate
accounts for the development of bacterial resistance to antibodies
Scientists believe that transposes, called retrotransposons represent degenerated retroviruses
retrotransposons are transported by being first transcribed into RNA
action of an enzyme produces DNA complementary to the RNA
constitutes as a copy of the original retrotransposon, becomes inverted into DNA
true
false
cDNA
A DNA sequence that is complementary to mRNA molecule
synthesized in vitro by an enzyme called reverse transcriptase
A population of individuals or DNA fragments that are identical
A DNA polymerase that uses an RNA template
Reverse transcriptase
DNA molecule used to direct the replication of a cloned DNA fragment in a host cell
A plasmid or other DNA sequences capable of self-replication
can be used for insertion of foreign DNA sequences
A DNA polymerase that uses an RNA template
Vector
A plasmid or other DNA sequences capable of self-replication
can be used for insertion of foreign DNA sequences
DNA molecule used to direct the replication of a cloned DNA fragment in a host cell
a small autonomously replicating circular double strand DNA present in bacteria and eukaryotes
Clone
A plasmid or other DNA sequences capable of self-replication
can be used for insertion of foreign DNA sequences
A population of individuals or DNA fragments that are identical
a small autonomously replicating circular double strand DNA present in bacteria and eukaryotes
Cloning vector
A population of individuals or DNA fragments that are identical
DNA molecule used to direct the replication of a cloned DNA fragment in a host cell
A plasmid or other DNA sequences capable of self-replication
can be used for insertion of foreign DNA sequences
Plasmid
a small autonomously replicating circular double strand DNA present in bacteria and eukaryotes
DNA molecule used to direct the replication of a cloned DNA fragment in a host cell
a fragment of DNA or RNA labeled with radioactivity, a dye, or an antigen
for detecting the presence of complementary sequences
Probe
a small autonomously replicating circular double strand DNA present in bacteria and eukaryotes
a fragment of DNA or RNA labeled with radioactivity, a dye, or an antigen
for detecting the presence of complementary sequences
forming a double stranded structure from 2 polynucleotide strands from different sources
Hybridization
forming a double stranded structure from 2 polynucleotide strands from different sources
use of radioactive or fluorescent probes to detect DNA or RNA sequences in cell extracts, chromosomes, or intact cells
uses antibodies to detect proteins separated by SDS-PAGE
In situ hybridization
use of radioactive or fluorescent probes to detect DNA or RNA sequences in cell extracts, chromosomes, or intact cells
forming a double stranded structure from 2 polynucleotide strands from different sources
mechanism by which bacteria transfers genes from one strain to another
occurs when DNA from a donor is added to medium and taken by the recipient
the recipient gene is known as transformant
Immunoblotting
method to separate proteins by gel electrophoresis on the basis of size
uses antibodies to detect proteins separated by SDS-PAGE
mechanism by which bacteria transfers genes from one strain to another
occurs when DNA from a donor is added to medium and taken by the recipient
the recipient gene is known as transformant
SDS-PAGE
method to separate proteins by gel electrophoresis on the basis of size
enzyme that cleaves DNA at a specific sequence
a virus for which the natural host is a bacteria cell
bacteria eaters
Restriction enzyme
enzyme that cleaves DNA at a specific sequence
mechanism by which bacteria transfers genes from one strain to another
occurs when DNA from a donor is added to medium and taken by the recipient
the recipient gene is known as transformant
joining of 2 DNA molecules with a covalent bond
Transformation
a virus for which the natural host is a bacteria cell
bacteria eaters
mechanism by which bacteria transfers genes from one strain to another
occurs when DNA from a donor is added to medium and taken by the recipient
the recipient gene is known as transformant
techniques for separating, uniting, and amplifying heterologous DNA molecules
bacteriophage
a virus for which the natural host is a bacteria cell
bacteria eaters
joining of 2 DNA molecules with a covalent bond
techniques for separating, uniting, and amplifying heterologous DNA molecules
Ligation
enzyme that cleaves DNA at a specific sequence
uses antibodies to detect proteins separated by SDS-PAGE
joining of 2 DNA molecules with a covalent bond
Recombinant DNA technology
A DNA insert joined to a vector
techniques for separating, uniting, and amplifying heterologous DNA molecules
exchange of genetic material
Recombinant molecule
A DNA insert joined to a vector
exchange of genetic material
techniques for separating, uniting, and amplifying heterologous DNA molecules
Recombination
A DNA insert joined to a vector
exchange of genetic material
a unit of DNA capable of replication (plasmid or chromosome)
Cosmid
a vector that contains bacteriophage lambda sequences, antibiotic resistance sequences and an origin of replication
can accommodate large DNA inserts
a unit of DNA capable of replication (plasmid or chromosome)
joining of 2 DNA molecules with a covalent bond
Replicon
A DNA insert joined to a vector
exchange of genetic material
a unit of DNA capable of replication (plasmid or chromosome)
Isopycnic centrifugation
Is a variant of the basic technique of ultracentrifugation Can be used to isolate DNA The densities of DNAs are about the same as concentrated solutions of cesium chloride
A technique where the components of a sample (DNA) are separated on the basis of their density in a centrifuge according to the centrifugal force they experience
Is an excellent means of removing proteins and RNA in the purification of DNA
The proteins float near the top of the tube The DNA bands near the center of the tube The RNA forms the pellets at the bottom of the tube
Density gradient centrifugation
Is a variant of the basic technique of ultracentrifugation Can be used to isolate DNA The densities of DNAs are about the same as concentrated solutions of cesium chloride
A technique where the components of a sample (DNA) are separated on the basis of their density in a centrifuge according to the centrifugal force they experience
Is an excellent means of removing proteins and RNA in the purification of DNA
Cesium chloride centrifugation
A technique where the components of a sample (DNA) are separated on the basis of their density in a centrifuge according to the centrifugal force they experience
Is an excellent means of removing proteins and RNA in the purification of DNA
The proteins float near the top of the tube The DNA bands near the center of the tube The RNA forms the pellets at the bottom of the tube
Is a variant of the basic technique of ultracentrifugation Can be used to isolate DNA The densities of DNAs are about the same as concentrated solutions of cesium chloride
DNA extraction Eukaryotes
Cell disruption (nuclei separation) Nuclei are treated with detergents and digestion with proteolytic enzymes and ribonuclease Extraction of DNA with organic solvents (in general a mixture of phenol-chloroform) Precipitation of DNA (ethanol) DNA dissolution in aqueous buffer Evaluation of DNA solution by spectrophotometry (UV)
Bacterial cells are disrupted by successive cycles of freezing-thawing (several times) Addition of ribonuclease enzyme and purified protinases to destroy RNA and proteins Density gradient centrifugation (cesium chloride)
DNA extraction prokaryotes
Bacterial cells are disrupted by successive cycles of freezing-thawing (several times) Addition of ribonuclease enzyme and purified protinases to destroy RNA and proteins Density gradient centrifugation (cesium chloride)
Cell disruption (nuclei separation) Nuclei are treated with detergents and digestion with proteolytic enzymes and ribonuclease Extraction of DNA with organic solvents (in general a mixture of phenol-chloroform) Precipitation of DNA (ethanol) DNA dissolution in aqueous buffer Evaluation of DNA solution by spectrophotometry (UV)
Characterization and measuring nucleic acid content
measured by spectrophometry
DNA and RNA
260
300
280
Characterization and measuring nucleic acid content
measured by spectrophometry
protein
280
260
300
220
Measurement of the nucleus acid content of whole cell or tissue homogenates requires chemical methods
true
false
DNA and RNA absorb at 260 due to
the conjugated double bonds present in their constituent bases
the unconjugated double bonds present in their constituent bases
the unconjugated single bonds present in their constituent bases
Spectrofluorimetry
Cleave some of the phosphodiester bonds Hydrolyse the glycosidic links between the deoxyribose and purines Deoxyribose residues are converted to hydroxylevulinyl aldehyde Hydroxylevulinyl aldehyde reacts with diphenylamine to produce a blue pigment The blue pigment is assayed at 600 nm
This is the ebay approach for samples where the DNA concentration is too low
The method uses the fluorescent dye ethidium bromide Ethidium bromide binds to dsDNA by insertion between stacked base pairs (intercalation)
The fluorescence of ethidium bromide is enhanced 25X when it interacts with dsDNA ssDNA gives no significant enhancement of fluorescence The conc of dsDNA can be calculated by comparing its fluorescence with that of standard dsDNA of known conc
A technique where the components of a sample (DNA) are separated on the basis of their density in a centrifuge according to the centrifugal force they experience
Chemical methods: DNA
Mostly based on colorimetric reactions with the Pentose groups of nucleic acids The total DNA concentration can be measured by the diphenylamine reaction Diphenylamine is specific for 2-deoxypentoses Diphenylamine reaction involves heating in a boiling water bath for 10 min: DNA solution Diphenylamine reagent in acetic acid Concentrated sulphuric acid
Orcinol reaction is a general assay for pentoses Orcinol reaction involves heating in a boiling bath RNA solution Orcinol reagent The orcinol reagent is prepared by dissolving 1g of FeCl36H2O in 1 liter of concentrated HCl +35 ml of 6% w/v Orcinol in ethanol
Chemical methods: DNA
The acids
Cleave some of the phosphodiester bonds Hydrolyse the glycosidic links between the deoxyribose and purines Deoxyribose residues are converted to hydroxylevulinyl aldehyde Hydroxylevulinyl aldehyde reacts with diphenylamine to produce a blue pigment The blue pigment is assayed at 600 nm
Cleave some of the phosphodiester bonds Hydrolyses the glycosidic links between the ribose and purines The hot acid also converts the tibose to furfural The furfural reacts with orcinol in presence of ferric ions to produce green coloured compounds Green coloured compounds are assayed at 660 nm
Chemical methods: RNA
Mostly based on colorimetric reactions with the Pentose groups of nucleic acids The total DNA concentration can be measured by the diphenylamine reaction Diphenylamine is specific for 2-deoxypentoses Diphenylamine reaction involves heating in a boiling water bath for 10 min: DNA solution Diphenylamine reagent in acetic acid Concentrated sulphuric acid
Orcinol reaction is a general assay for pentoses Orcinol reaction involves heating in a boiling bath RNA solution Orcinol reagent The orcinol reagent is prepared by dissolving 1g of FeCl36H2O in 1 liter of concentrated HCl +35 ml of 6% w/v Orcinol in ethanol
Chemical methods: RNA
The acid
Cleave some of the phosphodiester bonds Hydrolyses the glycosidic links between the ribose and purines The hot acid also converts the tibose to furfural The furfural reacts with orcinol in presence of ferric ions to produce green coloured compounds Green coloured compounds are assayed at 660 nm
Cleave some of the phosphodiester bonds Hydrolyse the glycosidic links between the deoxyribose and purines Deoxyribose residues are converted to hydroxylevulinyl aldehyde Hydroxylevulinyl aldehyde reacts with diphenylamine to produce a blue pigment The blue pigment is assayed at 600 nm
To have an absolute concentration :
Determination of inorganic phosphate (ip) concentration
We can produce ip by heating nucleic acid in presence of perchloric acid (hydrolysis) Phosphate determination Can not differentiate between RNA and DNA
Dry and weigh
Separation without denaturation Good method but expensive Only if little material (DNA or RNA)
To have an absolute concentration:
DNA precipitation
Dry and weigh
heating nucleic acid in presence of perchloric acid (hydrolysis)
Separation without denaturation
DNA-RNA separation
Density gradient (cesium chloride) centrifugation can be used to separate DNA-RNA
Separation without denaturation Good method but expensive Only if little material (DNA or RNA)
Material of the column: calcium phosphate gel (hydroxyapatite) Hydroxyapatite binds to double stranded nucleic acid more tenaciously than to single stranded molecules dsDNA can be effectively separated from ssDNA, RNA and protein contaminants
DNAse or RNAse treatment Pure material
DNA-RNA separation
Column chromatography
Separation without denaturation Good method but expensive Only if little material (DNA or RNA)
Material of the column: calcium phosphate gel (hydroxyapatite) Hydroxyapatite binds to double stranded nucleic acid more tenaciously than to single stranded molecules dsDNA can be effectively separated from ssDNA, RNA and protein contaminants By eluting the column with increasing concentration of phosphate buffer dsDNA binds to the column, while the rest of the samples passes through
Gel electrophoresis
Methods that denature the nucleic acids
DNAse or RNAse treatment Pure material
DNAse or RNAse treatment unPure material
RNAse treatment Pure material
Determination of molecular weight
Gel electrophoresis (relative method)
DNA molecules are negatively charged Migrating through an Agarose gel towards the anode (+) at a rate which is dependent upon molecular size
true
false
Determination of molecular weight:Gel electrophoresis:DNA
Migration from - to + Type of DNA influences the migration process Linear DNA and circular DNA with the same MW migrate differently
true
false
Determination of molecular weight :Gel electrophoresis : RNA
Under conditions used to separate dsDNA, RNA molecules tend to develop a primary structure And this leads to anomalous mobilities To eliminate RNA secondary structure, samples are pre-treated by heating in dilute formamide or glyoxal And electrophoresis is carried out in “denaturing gel” which include buffers containing formaldehyde
true
false
For higher molecular weight (MW)
Impossible to determine the MW by gel electrophoresis or sequencing
true
false
for MW less than 200kb
Density gradient (saccharose) centrifugation
electric microscopy (EM)
For MW greater than 200kb
electric microscopy (EM)
Density gradient (saccharose) centrifugation
Denaturation of DNA
Physicochemical properties of DNA
Hydrogen bonds between base pairs can be disrupted The 2 strands are no longer held together (the strands separate as individual random coils)
true
false
Denaturation of DNA is prompted by
Heat
Low salt conc
Extremes in pH (basic or acidic media)
Chemical agent: formamide, urea
cold
Low salt conc
Extremes in pH (basic or acidic media)
Chemical agent: formamide, urea
Heat
high salt conc
Extremes in pH (basic or acidic media)
Chemical agent: formamide, urea
The rise in absorbance coincides with strand separation
true
false
And the temperature at which one-half of a DNA sample is denatured is termed: melting temperature (Tm)
true
false
Tm value is higher for
G-C
A-T
A-U
Renaturation of DNA
if the denaturing conditions are removed
DNA solution is cooled gradually
pH is returned to neutrality
Denaturants are diluted out
Denatured DNA will renature to reform the duplex structure
DNA solution is cooled gradually
pH stays the same
Denaturants are diluted out
Denatured DNA will renature to reform the duplex structure
DNA solution is cooled gradually
pH is returned to neutrality
Denaturants remain
Denatured DNA will renature to reform the duplex structure
annealing is the process of cooling heated samples of DNA and Annealing is a powerful mean for comparing samples of DNA from different sources for their relatedness
true
false
Nucleic acid hybridization
Artificial hybrid duplexes may form if the DNA from one species is similar in nucleotide sequence to the DNA of the other About 25% of the DNA from human forms hybrids with mouse DNA Indicating that some of the genes (nucleotide sequences) in humans are very similar to those in mice
true
false
Nucleic acid hybridization
Is a uncommon employed procedure in molecular biology
1) it can reveal evolutionary relationships
2) it gives researchers the power to identify specific genes selectively against a vast background of irrelevant genetic material
true
false
Southern Blotting
used for detection of specific genes in cellular DNA
target nucleic acid is RNA
identifying and characterizing specific gene product
Southern Blotting applications
gene under investigation is expressed in a specific tissue or what level the gene is expressed
identifying and characterizing specific gene product
rape and murder investigations
Northern blotting
the target nucleic acid is DNA
the target nucleic acid is RNA
protein
northern blotting application
tool for identifying and characterizing specific gene product
This method is now proving decisive in court cases worldwide (rape and murder victim)
An investigator may not know whether a gene under investigation is expressed in a specific tissue or what level the gene is expressed
western blot
A particular DNA product can be located
A particular protein product can be located
A particular RNA product can be located
western blot applications
powerful tool for identifying and characterizing specific gene product
An investigator may not know whether a gene under investigation is expressed in a specific tissue or what level the gene is expressed
In oncology, targeting certain specific DNA sequences in lymphoma and leukemia
In situ hybridization
used to detect nucleic acid sequences present in intact cells
true
false
In situ hybridization applications
to identify expression of specific mRNAs in individual cells present in tissues containing a number of different cell types
can be used to show that insulin mRNA is produced only by the beta cells of the pancreas
The detection of specific pathogenic microbes and the detection of particular genes
Dot blotting or slot blotting
The dot blotting allows detection of a particular nucleic acid sequence in a sample
true
false
dot blotting or slot blotting applications
identify expression of specific mRNAs in individual cells present in tissues containing a number of different cell types
The detection of specific pathogenic microbes and the detection of particular genes
powerful tool for identifying and characterizing specific gene product
The probes used in blotting and DNA hybridization can be obtained from a variety of sources including:
cDNA
oligonucleotide probes
polymerase chain reaction
zDNA
oligonucleotide probes
polymerase chain reaction
cDNA
oligonucleotide probes
polymerase chain reaction
Heterologous probes
labeling of probes
Nick-translation technique
involves introducing single strand breaks (nicks) in the DNA
involve incorporating a modified nucleotide precursor, such as dTTP, into the DNA by nick-translation
Labeling of probes
enzyme-linked methods
involves introducing single strand breaks (nicks) in the DNA
involve incorporating a modified nucleotide precursor, such as dTTP, into the DNA by nick-translation
DNA fingerprinting
discovered by Alec Jefferys
human genome contains many microsatellite regions
repeated nucleotide sequences, all of which have a short common core sequence
discovered by Griffith
human genome contains many microsatellite regions
repeated nucleotide sequences, all of which have a short common core sequence
discovered by Alec Jefferys
human genome contains 1 microsatellite region
repeated nucleotide sequences, all of which have a short common core sequence
DNA fingerprints applications
biological evidence
personal identification
diagnosis of inherited disorders
biological evidence
personal identification
personal identification
DNA sequencing
(Sanger)
makes use of dideoxynucleotides, which have no -OH group at either C-2 or C-3 of ribose
A dideoxynucleoside triphosphate (ddNTP) can be added to a growing DNA chain
But since it lacks an -OH group at C-3 position it cannot form a phosphodiester bond with the next deoxynucleoside triphosphate (dNTP)
Dideoxynuleotide acts as a terminator at the site it occupies
This technique is based on chemical degradation using different reagents to break the target DNA sequence into fragments at each point where a particular base occurs
DNA sequencing (Maxam-Gilbert)
This technique is based on chemical degradation using different reagents to break the target DNA sequence into fragments at each point where a particular base occurs
makes use of dideoxynucleotides, which have no -OH group at either C-2 or C-3 of ribose
A dideoxynucleoside triphosphate (ddNTP) can be added to a growing DNA chain
But since it lacks an -OH group at C-3 position it cannot form a phosphodiester bond with the next deoxynucleoside triphosphate (dNTP)
Dideoxynuleotide acts as a terminator at the site it occupies
The polymerase chain reaction (PCR)
This technique for amplifying DNA sequences was developed by Mullis
This technique for amplifying DNA sequences was developed by Tullis
This technique for amplifying DNA sequences was developed by mark
The PCR can be used on DNA or RNA fragments that are present at extremely low concentrations
true
false
PCR is carried out at 3 stages with different temperature
denaturation: above 90
annealing: 40-60
extension: 72
denaturation: above 90
annealing: 40-60
extension: 80
denaturation: above 62
annealing: 90
extension: 72
taq polymerase is heat stable but does not possess proofreading properties
true
false
restriction enzymes
discovered by smith and nathans and arber
recognize and degrade DNA from foreign organisms
any DNA that enters the bacterial cell and is not methylated at those specific sites is subject to cleavage by restriction endonucleases
3 types
discovered by smith and nathans and arber
recognize and degrade DNA from foreign organisms
any DNA that enters the bacterial cell and is not methylated at those specific sites is subject to cleavage by restriction endonucleases
2 types
discovered by smith and nathans and arber
recognize and degrade DNA from foreign organisms
any DNA that enters the bacterial cell and is methylated at those specific sites is subject to cleavage by restriction endonucleases
3 types
restriction enzymes
type 1
requires ATP
cuts randomly
requires ATP
cuts at specific sites
cuts randomly
restriction enzymes
type 3
requires ATP
cuts at specific nucleotide site
cuts at specific nucleotide site
does not require ATP
cuts at specific nucleotide site
restriction enzyme
type 2
modify DNA by methylation
do not require ATP
cleave DNA at specific sites
reads the same forward or backward (palindromic)
ex refer
do not modify DNA by methylation
do not require ATP
cleave DNA at specific sites
reads the same forward or backward (palindromic)
ex refer
do not modify DNA by methylation
require ATP
cleave DNA at specific sites
reads the same forward or backward (palindromic)
ex refer
DNA restriction fragments having sticky ends can be joined together with DNA ligase to create new combinations of DNA sequences
true
false
restriction mapping
involves binding the DNA with one or more of a series of different restriction enzymes and separating the resulting fragments according to size by agarose gel electrophoresis
involves cutting the DNA with one or more of a series of different restriction enzymes and separating the resulting fragments according to size by agarose gel electrophoresis
Restriction fragment length polymorphisms (RFLP)
There are slight DNA sequence differences that occur from individual to individual
Once every 200 to 500 base pair
There are slight DNA sequence differences that occur from individual to individual
1-RFLP
used to identify individuals
used to screen for genetic diseases
2-RFLP
used to identify individuals
used to screen for genetic diseases
Nuclease S1, isolated from certain Neurospora and Aspergillus species This enzyme hydrolyzes single stranded DNA and RNA It is used to eliminate non-annealed polynucleotide tails and hair-pin loops in DNA RNA or DNA DNA duplexes in hybridization studies and in genetic recombination experiments
true
false
Deoxyribonuclease (DNAse I)
degrades randomly single-stranded DNA and double stranded DNA
No specific recognition site
This enzyme is used for limited digestion of DNA in a variety of molecular biology techniques,
degrades randomly single-stranded DNA and double stranded DNA
specific recognition site
This enzyme is used for limited digestion of DNA in a variety of molecular biology techniques,
degrades single-stranded DNA and double stranded DNA
No specific recognition site
This enzyme is used for limited digestion of DNA in a variety of molecular biology techniques,
RNA sequencing
When RNA is to be sequenced, the method of choice is not to analyze RNA itself but to use the methods of DNA sequencing on a DNA complementary (cDNA) to the RNA in question The cDNA in turn is generated by using the enzyme reverse transcriptase The enzyme transcriptase catalyzes the synthesis of DNA from an RNA template
true
false
DNA cloning
3 categories
isolation
preparation
transfer
preparation
transfer
deletion
preparation
transfer
addition
selection and detection
These genes do not act as markers for the vector One gene (e.g. amp) can be used to select for bacteria which form colonies on an agar base media containing the antibiotic, while non-transformed (amp-sensitive) cells would be killed The other gene (e.g. tet) can be used as not a marker for the recombinant plasmid vector
true
false
cloning vector
are short molecules
cut at multiple positions
must be replicated in host cell
do not include markers
are long molecules
must be replicated in host cell
carry markers
short molecules
cut at only one position
must be replicated in host cell
must carry markers
DNA cloning
plasmids
simplest cloning vectors
capable of autonomous replication
most complicated cloning vectors
DNA cloning
phages
offer several disadvantages over plasmid vectors
carry larger fragments of DNA
offer several advantages over plasmid vectors
carry larger fragments of DNA
DNA cloning
Cosmids
must possess a 14kb sequence (cos) at each of its ends
must be separate by no more than 51 and no less than 36
reproduce in host bacteria as plasmids
true
false
Transgenic animals brinster and palmiter
introduced a gene for rat growth hormone
increase = gigantism
decrease = dwarfism
true
false
procedures for producing transgenic mammals
microinjection
Cells from the blastocyst stage of early mouse embryos can be removed and grown in culture These are called embryonic stem (ES) cells and have the ability to differentiate into all other cell types ES can be genetically modified in the laboratory and returned to the blastocysts for implantation
The injected eggs are then introduced into the oviduct of a recipient female or foster mother to develop This process is technically demanding and has a low success rate This is now the most widely used methods of producing transgenic animals
The process of inactivating a specific gene in a mouse is called gene targeting
procedures for producing transgenic mammals
embryonic stem cells
Cells from the blastocyst stage of early mouse embryos can be removed and grown in culture These are called embryonic stem (ES) cells and have the ability to differentiate into all other cell types ES can be genetically modified in the laboratory and returned to the blastocysts for implantation
The injected eggs are then introduced into the oviduct of a recipient female or foster mother to develop This process is technically demanding and has a low success rate This is now the most widely used methods of producing transgenic animals
The process of inactivating a specific gene in a mouse is called gene targeting
Procedures for producing transgenic mammals
creating knockout mice
The injected eggs are then introduced into the oviduct of a recipient female or foster mother to develop This process is technically demanding and has a low success rate This is now the most widely used methods of producing transgenic animals
The process of inactivating a specific gene in a mouse is called gene targeting,
Cells from the blastocyst stage of early mouse embryos can be removed and grown in culture These are called embryonic stem (ES) cells and have the ability to differentiate into all other cell types ES can be genetically modified in the laboratory and returned to the blastocysts for implantation
Procedure for producing transgenic plants
microprojectile bombardment:
involves shooting DNA coated tungsten or gold particles into plant cells
The Ti plasmid can be isolated from bacteria and linked with foreign genes to produce a recombinant plasmid that is taken up in culture by plant cells
uses a short burst of electricity to get the DNA into the cell
Procedure for producing transgenic plants
electroporation
involves shooting DNA coated tungsten or gold particles into plant cells
uses a short burst of electricity to get the DNA into the cell
During an infection, the Ti plasmid is passed from bacterium into the plant cell where it becomes incorporated into plant cell’s chromosome The Ti plasmid can be isolated from bacteria and linked with foreign genes to produce a recombinant plasmid that is taken up in culture by plant cells
Procedure for producing transgenic plants
agrobacterium tumefaciens-mediated transformation
During an infection, the Ti plasmid is passed from bacterium into the plant cell where it becomes incorporated into plant cell’s chromosome The Ti plasmid can be isolated from bacteria and linked with foreign genes to produce a recombinant plasmid that is taken up in culture by plant cells
involves shooting DNA coated tungsten or gold particles into plant cells
uses a short burst of electricity to get the DNA into the cell
Gene therapy
Gene therapy is the attempt to treat and possibly cure human disease, including genetic defects, cancer, diabetes etc.. with cloned genes
true
false
Transcription in prokaryotes
Francis crick enunciated the central dogma of molecular biology
The transcription requires an enzyme known as DNA-dependent RNA polymerase (RNA polymerase)
A distinguishing feature of RNA polymerase is its ability to initiate chain growth without the need of a primer, unlike DNA polymerase
true
false
Transcription Regulation in Prokaryotes
operons regulate
operons do not regulate
