Ch. 6: DNA and Biotechnology Flashcards
1
Q
what are the two distinct forms of nucleic acids within eukaryotic cells?
A
- DNA (deoxyribonucleic acid)
- RNA (ribonucleic acid
2
Q
in sum: what are DNA and RNA
A
polymers with distinct roles that together create the molecules integral to life in al living organisms
3
Q
where is the bulk of DNA found? where are the other 2 less common locations?
A
in chromosomes in the nucleus of eukaryotic cells
some also present in mitochondria and chloroplasts
4
Q
explain what it means that DNA is a macromolecule
A
DNA is a polydeoxyribonucleotide that is composed of many monodeoxyribonucleotides linked together
5
Q
defn: nucleosides
A
composed of a 5-C sugar (pentose) bonded to a nitrogenous base and are formed by covalently linking the base to C-1’ of the sugar
6
Q
what does the ‘ in nucleoside notation mean?
A
the C atoms in the SUGAR are labeled with a ‘ to distinguish them from the C atoms in the nitrogenous base
7
Q
defn: nucleotides
A
formed when one or more phosphate groups are attached to C-5’ of a nucleoside
the building blocks of DNA
8
Q
what are nucleotides named by? + examples
A
according to the number of phosphates present
adenosine di- and triphosphate (ADP and ATP) gain their names from the number of phosphate groups attached to the nucleoside adenosine
9
Q
why are nucleotides high-energy compounds
A
because of the energy associated with the repulsion between closely associated negative charges on the phosphate groups
10
Q
what are nucleic acids classified according to?
A
the pentose they contain
11
Q
if the pentose is a RIBOSE, what is the nucleic acid?
if the pentose is a DEOXYRIBOSE, what is the nucleic acid?
A
ribose –> RNA
deoxyribose –> DNA
12
Q
defn + diagram: ribose vs. deoxyribose
A
deoxyribose is ribose with the 2’-OH group replaced by -H
13
Q
nomenclature: base, nucleoside, and nucleotides corresponding
A
14
Q
what is the backbone of DNA composed of? + func + how is it formed
A
composed of: alternating sugar and phosphate groups
func: determines the directionality of the DNA, is always read from 5’ to 3’
formed: as nucleotides are joined by 3’-5’ phosphodiester bonds (a phosphate group links the 3’ C of one sugar to the 5’ phosphate group of the next incoming sugar in the chain)
15
Q
why do DNA and RNA strands have an overall negative charge?
A
because phosphates carry a negative charge
16
Q
why is their polarity within the backbone of DNA?
A
each strand of DNA has distinct 5’ and 3’ ends
the 5’ end will have an -OH or phosphate group bonded to C-5’ of the sugar
the 3’ end has a free -OH on C-3’ of the sugar
17
Q
is the base sequence of a nucleic acid strand written or read in the 5’ to 3’ direction?
A
both written and read
18
Q
how is the DNA strand written? (4 ways)
A
main way: 5’-ATG-3’ (simply ATG)
backwards: 3’-GTA-5’
showing phosphates: pApTpG
using “d” as shorthand for deoxyribose: dAdTdG
19
Q
defn: dsDNA, ssRNA
A
the general forms of DNA and RNA
dsDNA = double-stranded DNA
ssRNA = single-stranded RNA
20
Q
group + char (2): purines and pyrimidines
A
the two families of nitrogen-containing bases
biological aromatic heterocycles
exceptional stability
21
Q
struct + what are they + in DNA or RNA: purines
A
contain 2 rings in their structure
adenine (A) and guanine (G) both of which are found in DNA and RNA
22
Q
struct + what are they + in DNA or RNA: pyrimidines
A
contain 1 ring in their structure
cytosine (C), thymine (T), uracil (U)
cytosine: DNA and RNA
thymine: DNA
uracil: RNA
23
Q
mnemonic: purines and pyrimidines
A
PURe As Gold (A and G are purines)
it takes 2 gold rings at a wedding, just like purines had 2 rings in their structure
24
Q
what 4 rules do aromatic compounds follow in chemistry? + defn aromatic
A
defn: any unusually stable ring system that adheres to these 4 rules
- the compound is cyclic
- the compound is planar
- the compound is conjugated (has alternating single and multiple bonds, or lone pairs, creating at least one unhybridized p-orbital for each atom in the ring)
- the compound has 4n+2 (where n is any integer) pi electron = Huckel’s rule
25
what is the most common example of an aromatic compound?
benzene
26
what is the extra stability due to in an aromatic compound?
delocalized pi electrons, which can travel throughout the entire compound using available molecular orbitals
27
molecular char (3) + diagram: benzene
1. all 6 of the C atoms are sp2 hybridized
2. each of the 6 orbitals overlaps equally with its 2 neighbors
3. so, the delocalized electrons form 2 pi electron clouds (one above and one below the plane of the ring)
28
why aromatic molecules are fairly unreactive?
the delocalization of electrons to form electron clouds
29
defn: heterocycles
ring structures that contain at least 2 different elements in the ring
30
why, in part, are nucleotides so useful as the molecule to store genetic info?
they have exceptional stability!
31
4 key features: Watson-Crick model of DNA structure
1. the 2 strands of DNA are antiparallel
2. the sugar-phosphate backbone is on the outside of the helix with the nitrogenous bases on the inside
3. there are specific base-pairing rules (complementary base-pairing)
4. because of the specific base-pairing, the amount of A equals the amount of T, and the amount of G equals the amount of C THUS total purines = total pyrimidines overall --> Chargaff's rules
32
explain: the two strands of DNA are antiparallel
the strands are oriented in opposite directions
when one strand has polarity 5' to 3' down the page, the other stand has 5' to 3' polarity up the page
33
explain (5) + diagram: complementary-base pairing
1. a adenine (A) is always base-paired with a thymine (T) via two H bonds
2. a guanine (G) always pairs with cytosine (C) via 3 H bonds
3. the 3 H bonds make the G-C base pair interaction stronger
4. these H bonds, and the hydrophobic interactions between bases, provide stability to the double helix structure
5. the base sequence on one strand defines the base sequence on the other strand
34
structure: double helix
two linear polynucleotide chains of DNA are wound together in a spiral orientation along a common axis
35
defn + char (4) + diagram: B-DNA
the double helix of most DNA
1. a right-handed helix
2. makes a turn every 3.4 nm
3. contains about 10 bases within that span
4. major and minor grooves can be identified between the interlocking strands and are often the site of protein binding
36
defn + char (7): Z-DNA
another form of DNA
1. zigzag appearance
2. left-handed helix
3. has a turn every 4.6 nm
4. contains 12 bases within each turn
5. may arise from a high GC-content or a high salt concentration
6. not associated with any biological activity
7. unstable
37
func + process: denaturation
a way of gaining access to DNA during processes such as replication and transcription
the double helical nature of DNA can be denatured by conditions that disrupt hydrogen bonding and base-pairing, resulting in the "melting" of the double helix into 2 single strands that have separated from each other
38
during denaturation, do the covalent links between nucleotides in the DNA backbone break?
No
39
what 3 things are commonly used to denature DNA?
1. heat
2. alkaline pH
3. chemicals like formaldehyde and urea
40
defn: reannealed
brought back together
41
when can denature, single-stranded DNA be reannealed? + example
if the denaturing condition is slowly removed
ex: if a solution of heat-denatured DNA is slowly cooled
42
diagram: denaturation + reannealing of DNA
43
defn: probe DNA
DNA with known sequence
44
how is DNA divided up in the cell?
among the 46 chromosomes found in the nucleus
45
defn: histones
a group of small basic proteins that the DNA that makes up a chromosome is wound around
46
defn: chromatin
histones + the DNA wrapped around them
47
how many histone proteins are found in eukaryotic cells?
5
48
what forms the histone core?
two copies of each of the histone proteins H2A, H2B, H3, and H4
49
defn + analogy + diagram: nucleosome
200 base pairs of DNA wrapped around this protein complex
analogy: beads on a string
50
func: H1
the last histone
seals off the DNA as it enters and leaves the nucleosome, adding stability to the structure
51
what to the nucleosomes create together?
a much more organized and compacted DNA
52
defn + ex + char (2): nucleoproteins
proteins that associate with DNA
ex: histones
char of most others: 1. acid soluble
2. tend to stimulate processes such as transcription
53
why is it helpful for DNA to be uncondensed when the cell undergoes DNA replication?
it makes it more accessible to make the process more efficient
54
defn + char (3): heterochromatin
a small percentage of the chromatin that remains compacted during interphase
char: 1. appears dark under light microscopy
2. transcriptionally silent
3. often consists of DNA with highly repetitive sequences
55
defn + char (2): euchromatin
the dispersed chromatin
char: 1. appears light under light microscopy
2. contains genetically active DNA (expressed)
56
summary + diagram: heterochromatin vs. euchromatin
heterochromatin = dark, dense, and silent
euchromatin = light, uncondensed, and expressed
57
why can't DNA replication extend all the way to the end of a chromosome?
this will result in losing sequences and information with each round of replication
58
defn + func: telomere
a simple repeating unit (TTAGGG) at the end of the DNA
func: 1. the solution for our cells for the fact that DNA replication can't extend all the way to the end of a chromosome
2. their high GC-content creates exceptionally strong strand attractions at the end of chromosomes to prevent unraveling (think of telomeres as "knotting off" the end of the chromosome)
59
func: telomerase
the enzyme that replaces some of the sequence that is lost in each round of replication
60
is telomerase more or less expressed in rapidly dividing cells?
more highly expressed
61
what does the progressive shortening of telomeres contribute to? why does this happen?
contributes to aging
there are a set number of replications possible
62
defn + func + char (2): centromeres
a region of DNA found in the center of chromosomes
char: 1. often referred to as sites of constriction because they form noticeable indentations
2. composed of heterochromatin which is composed of tandem repeat sequences that also contain high GC-content
func: during cell division, the two sister chromatids can thus remain connected at the centromere until microtubules separate the chromatids during anaphase
63
analogy: DNA
an organism's blueprint
64
func: DNA
provides the ability to sustain activities of life and insight into our evolutionary past
65
func: DNA replication
necessary for reproduction of a species and for any dividing cell
66
defn + aka: replisome
aka: replication complex
a set of specialized proteins that assist the DNA polymerases
67
defn + func + diagram: origins of replication
points at which DNA unwinds to begin the process of replication
68
cause + effect: replication forks
the generation of new DNA proceeds in both directions, which creates replication forks on both sides of the origin
increases the efficiency of replication
69
char + behavior in replication: bacterial chromosome (3)
1. closed, double-stranded circular DNA molecule
2. single origin of replication
3. two replication forks that move away from each other in opposite directions around the circle (these eventually meet, resulting in the production of two identical circular molecules of DNA)
70
why is eukaryotic replication slower than prokaryotic replication?
eukaryotic replication must copy many more bases
71
what does each eukaryotic chromosome contain in order to duplicate all of the chromosomes efficiently?
each contains one linear molecule of double-stranded DNA having multiple origins of replication
72
role in replication: sister chromatids vs. centromere
as the replication forks move toward each other and sister chromatids are created, the chromatids will remain connected at the centromere
73
defn + effect(2): helicase
the enzyme responsible for unwinding the DNA
1. generating two single-stranded template strands ahead of the polymerase
2. causes positive supercoiling that strains the DNA helix
74
what does it mean that the unpaired strands of DNA are very sticky once opened?
the free purines and pyrmidines seek out other molecules with which to hydrogen bond
75
func: single-stranded DNA-binding proteins
bind to the unraveled strand, preventing both the reassociation of the DNA strands and the degradation of DNA by nucleases
76
defn + analogy: supercoiling
a wrapping of DNA on itself as its helical structure is pushed ever further toward the telomeres during replication
analogy: an old-fashioned telephone cord that's tangled on itself
77
func: DNA topoisomerases
introduce negative supercoils to alleviate the torsional stress caused by positive supercoiling and to reduce the risk of strand breakage
78
HOW do DNA topoisomerases carry out their function?
they work ahead of helicase, nicking one or both strands, allowing relaxation of the torsional pressure, and resealing the cut strands
79
func in replication: parental strands
serve as templates for the generation of new daughter strands
80
why is replication semiconservative?
because one parental strand is retained in each of the two resulting identical double-stranded DNA molecules
81
func: DNA polymerases
responsible for reading the DNA template (parental strand) and synthesizing the new daughter strand
82
direction + outcome: DNA polymerase
READS the template strand in a 3' to 5' direction
SYNTHESIZES the complementary strand in the 5' to 3' direction
results in a new double helix of DNA that has the required antiparallel orientation
83
at each replication fork, what direction is each strand oriented?
one is oriented in the correct direction for DNA polymerase
the other is antiparallel
84
with the exception of DNA polymerase's reading direction, everything in molecular biology is 5' to 3', what does this include? (4)
1. DNA synthesis
2. DNA repair
3. RNA transcription
4. RNA translation
85
defn + char (2): leading strand (at each replication fork)
the strand that is copied in a continuous fashion, in the same direction as the advancing replication fork
char: 1. will be read 3' to 5'
2. its complement will be synthesized in a ' to 3'
86
defn: lagging strand
the strand that is copied in a direction opposite the direction of the replication fork
87
why can't DNA polymerase read and synthesize on the lagging strand?
the parental strand has 5' to 3' polarity
88
how does it solve the fact that DNA polymerase can't read the lagging strand as it is?
small strands called Okazaki fragments are produced
as the replication fork continues to move forward, it clears additional space that DNA polymerase must fill in
each time DNA polymerase completes an okazaki fragment, it turns around to find another gap that needs to be filled in
89
why is the first step of DNA replication to lay down an RNA primer? (2)
1. DNA cannot be synthesized de novo, so it needs another molecule to "hook on" to (however, RNA can be directly paired with the parent strand)
2. so, primase synthesizes a short primer (~10 nucleotides) in the 5' to 3' direction to start replication on each strand
90
how many primers does the leading and lagging strand require?
LAGGING: the primers (short RNA sequences) are constantly being added to the lagging strand because each Okazaki fragment must start with a new primer
LEADING: requires only one, in theory (reality: usually a few primers on the leading strand)
91
which DNA polymerases correspond to prokaryotes and which correspond to eukaryotes?
prokaryotes: DNA polymerase III
eukaryotes: DNA polymerases α, δ, and ε
92
steps (6): DNA replication
1. lay down an RNA primer
2. DNA polymerases begin synthesizing the daughter strands of DNA in the 5' to 3' manner
3. as the new phosphodiester bond is made, a free pyrophosphate (PPi) is released
4. the RNA is removed to maintain integrity of the genome
5. DNA polymerase I (prokaryotes) and DNA polymerase δ (eukaryotes) adds DNA nucleotides where the RNA primer had been
6. DNA ligase seals the ends of the DNA molecules together, creating one continuous strand of DNA
93
what are the incoming nucleotides in DNA replicaton?
5' deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP)
94
how is the RNA eventually removed?
by the enzyme DNA polymerase I (prokaryotes) or RNase H (eukaryotes)
95
roles of the five "classic" DNA polymerases in eukaryotic cells: α, β, γ, δ, and ε (5)
1. α, δ, and ε work together to synthesize both the leading and lagging strands
2. δ also fills in the gaps left behind when RNA primers are removed
3. γ replicates mitochondrial DNA
5. δ and ε are assisted by the PCNA protein, which assembles into a trimer to form the sliding clamp which helps to strengthen the interaction between these DNA polymerases and the template strand
96
why does the chromosome become a little shorter each time DNA synthesis is carried out?
because DNA polymerase cannot complete synthesis of the 5' end of the starnd
97
func: telomeres
to lengthen the time that cells can replicate and synthesize DNA before necessary genes are damaged
98
what does the repetitive nature of telomeres allow for?
they can be slightly degraded between replication cycles without loss of function
99
what are the 3 main categories of DNA damage?
1. breaking of the DNA backbone
2. structural or spontaneous alterations of bases
3. incorporation of the incorrect base during replication
100
why are cancer cells able to proliferate excessively?
because they can divide without stimulation from other cells and are no longer subject to the normal controls on cell proliferation
101
what are the 2 ways that cancer cells can migrate?
1. local invasion
2. metastasis
102
defn: metastasis
a migration to distant tissues by the bloodstream or lymphatic system
103
over time, do cancer cells accumulate mutations or does the number of mutations stay constant?
they accumulate mutations
104
defn: allele
one of two or more alternative forms of a gene that arise by mutation and are found at the same place on a chromosome
105
defn + func + char (2): oncogenes
mutated genes that cause cancer
func: primarily encode cell cycle-related proteins
char: 1. the abnormal alleles (mutated genes) encode proteins that are more active than normal proteins, promoting rapid cell cycle development
2. a mutation in only one copy is sufficient to promote tumor growth and is thus considered dominant
106
defn: proto-oncogenes
oncogenes before they are mutated
107
what was the first oncogene to be discovered?
src (sarcoma)
108
func (3) + ex (2) + aka: tumor suppressor genes
func: 1. encode proteins that inhibit the cell cycle or participate in DNA repair processes
2. function to stop tumor progression
3. mutations of these genes result in the loss of tumor suppression activity, and thus promote cancer
aka: antioncogenes
ex: p53, Rb (retinoblastoma)
109
why is inactivation of both alleles (mutated tumor suppressor genes) necessary for loss of function of tumor suppression activity?
in most cases, even one copy of the normal protein (non-mutated tumor suppression gene) can function to inhibit tumor formation (so multiple mutations are required)
110
oncogenes vs. mutated tumor suppressor genes: common and different + analogy
SAME = outcome = cancer
DIFFERENT = cause
1. oncogenes: promote the cell cycle (stepping on the gas)
2. mutated tumor suppressor genes: can no longer slow the cell cycle (cutting the brakes)
111
process (3) + diagram: proofreading
1. during synthesis the two double-stranded DNA molecules will pass through a part of the DNA polymerase enzyme for proofreading
2. when the complementary strands have incorrectly paired bases, the hydrogen bonds between the strands can be unstable and this lack of stability is detected as the DNA passes through this part of the polymerase
3. the incorrect base is excised and can be replaced with the correct one
112
if both the parent and daughter strands are simply DNA, how does the enzyme discriminate which is the template strand and which is the incorrectly paired daughter strand?
it looks at the level of methylation
the template strand has existed in the cell for a longer period of time and therefore is more methylated
113
why is the likelihood of mutations in the lagging strand considerably higher than the leading strand?
the DNA polymerase enzyme proofreading system is very efficient, correcting most of the errors put into the sequence during replication
however, DNA ligase, which closes the gaps between Okazaki fragments, lacks proofreading ability
114
char (2): mismatch repair
1. machinery for this is in the G2 phase of the cell cycle
2. enzymes for this encoded by genes MSH2 and MLH1 which detect and remove errors introduced in replication that were missed during the S phase of the cell cycle
115
what are the prokaryotic homologues of the enzymes coded by MSH2 and MLH1 mismatch repair genes?
MutS and MutL
116
how do most DNA repair mechanisms work (3 steps)?
1. involve proteins that recognize damage or a lesion
2. remove the damage
3. and then use the complementary strand as a template to fill in the gap
117
how does UV light affect DNA replication?
1. UV light induces the formation of dimers between adjacent thymine residues in DNA
2. the formation of thymine dimers interferes with DNA replication and normal gene expression and distorts the shape of the double helix
118
defn + process (4) + func + diagram: nucleotide excision repair (NER)
defn: a cut-and-patch process
func: eliminate thymine dimers from DNA
process: 1. specific proteins scan the DNA molecule and recognize the lesion because of a bulge in the strand
2. an excision endonuclease then makes nicks in the phosphodiester backbone of the damaged strand on both sides of the thymine dimer and removes the defective oligonucleotide
3. DNA polymerase can then fill in the gap by synthesizing DNA in the 5' to 3' direction, using the undamaged strand as a template
4. the nick in the strand is sealed by DNA ligase
119
what happens when thermal energy is absorbed by DNA? (2)
1. this may lead to cytosine deamination (the loss of an amino group from cytosine and results in the conversion of cytosine to uracil)
2. uracil should not be found in a DNA molecule and is thus easily detected as an error
120
process (3) + func: base excision repair
to fix small, non-helix-distorting mutations
1. the affected base is recognized and removed by a glycosylase enzyme, leaving behind an apurinic/apyrimidinic (AP) site (aka an abasic site)
2. the AP site is recognized by an AP endonuclease that removes the damaged sequence from the DNA
3. DNA polymerase and DNA ligase can then fill in the gap and seal the strand
121
func (4): Recombinant DNA technology
1. allows a DNA fragment from any source to be multiplied by either gene cloning or PCR, providing a means of analyzing and altering genes and proteins
2. provides the reagents necessary for genetic testing (carrier detection, prenatal diagnosis)
3. useful for gene therapy
4. can provide a source of a specific protein
122
defn + func + char: DNA cloning
defn: a technique that can produce large amounts of a desired sequence
char: the DNA to be cloned is present in a small quantity and is part of a heterogenous mixture containing other DAN sequences
func: to produce a large quantity of homogenous DNA for other applications
123
what are vectors usually?
bacterial or viral plasmids that can be transferred to a host bacterium after insertion of the DNA of interest
124
process (5): DNA cloning
1. requires that the investigator ligate the DNA of interest into a piece of nucleic acid (a vector), forming a recombinant vector
2. the bacteria are then grown in colonies and a colony containing the recombinant vector is isolated, this can be accomplished by ensuring that the recombinant vector also includes a gene for antibiotic resistance
3. antibiotics can then kill off all of the colonies that do not contain the recombinant vector
4. the resulting colony can then be grown in large quantities
5. depending on the goal, the bacteria can then be made to express the gene of interest (generating large quantities of recombinant protein) or can be lysed to reisolate the replicated recombinant vectors (which can be processed by restriction enzymes to release the cloned DNA from the vector)
125
defn + aka + char + func: restriction enzymes
aka: restriction endonucleases
enzymes that recognize specific double-stranded DNA sequences which are palindromic (the 5' to 3' sequence of one strand is identical to the 5' to 3' sequence of the other strand in an antiparallel orientation)
char: isolated from bacteria (their natural source)
func: once a specific sequence has been identified, the restriction enzyme can cut through the backbones of the double helix
126
some restriction enzymes produce offset cuts, yielding sticky ends on the fragments, what are sticky ends advantageous in? + diagram
facilitating the recombination of a restriction fragment with the vector DNA
127
what allows the restriction fragment to be inserted directly into the vector?
the vector of choice can be cut with the same restriction enzyme
128
what does a vector require to allow for selection of colonies with recombinant plasmids? (2)
an origin of replication
at least one gene for antibiotic resistance
129
defn: DNA libraries
large collections of known DNA sequences
130
what do DNA libraries equate to in sum? what are DNA libraries produced from?
produced from: DNA cloning
in sum: could equate to the genome of an organism
131
how are DNA libraries made?
DNA fragments, often digested randomly, are cloned into vectors and can be used for further study
132
do libraries consist of genomic DNA or cDNA?
either
133
defn: genomic libraries
contain large fragments of DNA and include both coding (exon) and noncoding (intron) regions of the genome
134
defn + char(2) + aka: cDNA libraries
complementary DNA libraries
constructed by reverse-transcribing processed mRNA
char: 1. lack noncoding regions such as introns
2. only includes the genes that are expressed in the tissue from which the mRNA was isolated
aka: expression libraries
135
why can only cDNA libraries be used to reliably sequence specific genes and identify disease-causing mutations, produce recombinant proteins, or produce transgenic animals?
genomic libraries contain the entire genome of an organism, but genes may by chance be split into multiple vectors
136
what are 3 examples of recombinant proteins?
1. insulin
2. clotting factors
3. vaccines
137
defn + 2 techniques: hybridization
the joining of complementary base pair sequences (uses two single-stranded sequences)
1. DNA-DNA recognition
2. DNA-RNA recognition
138
what 2 techniques is hybridization vital to?
1. PCR
2. Southern blotting
139
defn + background process: PCR
polymerase chain reaction
an automate process that can produce millions of copies of a DNA sequence without amplifying the DNA in bacteria
knowing the sequences that flank the desired region of DNA allows for the amplification of the sequence in between
140
what are the 4 things that a PCR reaction requires?
1. primers that are complementary to the DNA that flanks the region of interest (has high GC content 40-60% is optimal, as the extra H bonds between G and C add stability)
2. nucleotides (dATP, dTTP, dCTP, and dGTP)
3. DNA polymerase
4. heat (to cause the DNA double helix to melt apart/denature)
141
since the DNA polymerase found in humans doesn't work at high temps, what is used instead?
the DNA polymerase from Thermus aquaticus, a bacteria that thrives in the hot springs at Yellowstone (70 deg C)
142
process (2): PCR
1. the DNA of interest is denatured, replicated, and then cooled to allow reannealing of the daughter strands with the parent strands
2. this process is repeated several times, doubling the amount of DNA with each cycle, until enough copies of the DNA sequence are available for further testing
143
defn: gel electrophoresis
a technique used to separate macromolecules, such as DNA and proteins, by size and charge
144
explain how gel electrophoresis works for separating DNA (2)
1. all molecules of DNA are negatively charged because of the phosphate groups in the backbone, so all DNA strands will migrate toward the anode of an electrochemical cell
2. the preferred gel for DNA electrophoresis is agarose gel and the longer the DNA strand, the slower it will migrate in the gel
145
func: Southern blot
used to detect the presence and quantity of various DNA strands in a sample
146
process (4): Southern blotting
1. DNA is cut by restriction enzymes and then separated by gel electrophoresis
2 the DNA fragments are then carefully transferred to a membrane, retaining their separation
3. the membrane is then probed with many copies of a single-stranded DNA sequence
4. the probe will bind to its complementary sequence and form double-stranded DNA
147
what are probes labeled with? (2)
1. radioisotopes
2. indicator proteins
both of which can be used to indicate the presence of a desired sequence
148
what 5 things does a basic sequencing reaction contain?
1. template DNA
2. primers
3. an appropriate DNA polymerase
4. all 4 deoxyribonucleotide triphosphates
5. a modified base = a dideoxyribonucleotide in lower concentratons
149
how are dideoxyribonucleotides different thatn deoxyribonucleotides?
dideoxyribonucleotides = ddATP, ddCTP, ddGTP, ddTTP
contain a H at C-3', rather than a hydroxyl group
150
process (4): DNA sequencing
1. once one of the modified bases has been incorporated, the polymerase can no longer add to the chain
2. eventually the sample will contain many fragments (as many as the number of nucleotides in the desired sequence), each one of which terminates with one of the modified bases
3. these fragments are then separated by size using gel electrophoresis
4. the last base for each fragment can be read, and because gel electrophoresis separates the strands by size, the bases can easily be read in order
151
func + process (2): gene therapy
func: offers potential cures for individuals with inherited diseases
process: 1. intended for diseases in which a given gene is mutated or inactive, giving rise to pathology
2. by transferring a normal copy of the gene into the affected tissues, the pathology should be fixed, essentially curing the individual
152
what must be true for gene replacement therapy to be a realistic possibility?
efficient gene delivery vectors must be used to transfer the cloned gene into the target cells' DNA
153
what are most gene delivery vectors in use? why? how?
modified viruses
because viruses naturally infect cells to insert their own genetic material
a portion of the viral genome is replaced with the cloned gene such that the virus can infect but not complete its replication cycle
154
what is a risk of randomly integrated DNA?
there is a risk of integrating near and activating a host oncogene
155
defn: transgenic mice
altered at their germ line by introducing a cloned gene into fertilized ova or into embryonic stem cells
156
defn: transgene + what must be true about the transgene to be useful?
the cloned gene that is introduced into transgenic mice
if the transgene is a disease-producing allele, the transgenic mice can be used to study the disease process from early embryonic development throughout adulthood
157
defn + use: knockout mice
a gene has been intentionally deleted (knocked out)
valuable models in which to study human diseases
158
explain what happens when a cloned gene may be microinjected into the nucleus of a newly fertilized ovum (4) + diagram
1. the gene may then incorporate into the nuclear DNA of the zygote
2. the ovum is then implanted into a surrogate
3. if successful, the resulting offspring will contain the transgene in all of their cells, including their germ line cells (gametes)
4. the transgene will also be passed to THEIR offspring
159
what are transgenic mice useful for studying? what are they not useful for studying?
useful: dominant gene effects
not useful: model for recessive disease because the # of copies of the gene that insert into the genome cannot be controlled
160
how can embryonic stem cell lines be used for developing transgenic mice? (3) what are 2 advantages to this method?
advantage: 1. the cloned genes can be introduced in cultures
2. one can select for cells with the transgene successfully inserted
process: 1. the altered stem cells are injected into developing blastocysts and implanted into surrogates
2. the blastocyst itself is thus composed of 2 types of stem cells (ones containing the transgene and the original blastocyst cells that lack the transgene)
3. the resulting offspring is a chimera
161
defn + how to identify: chimera
has patches of cells, including germ cells, derived from each of the two lineages
evident if: the two cell lineages (transgenic cells and host blastocyst) come from mice with different coat colors --> they will have patchy coats of two colors --> easy identification
162
what kind of offspring are chimeras able to produce? (2)
1. heterozygous for transgene
2. homozygous for transgene
163
what are 2 general safety and ethical issues that these topics bring rise to?
1. pathogen resistance
2. the ethics of choosing individuals for specific traits
