chapter 12 Flashcards
What is replication?
Cells can make exact copies of their DNA
what’s the name given to errors that are caused during replication?
mutations
How does replication happen? Explain
- two strands of parental DNA separate
- each serves as the template for
synthesis of a daughter strand - Replication occurs from 5’ to 3’
What are the different hypothetical models
for replication
semiconservative
conservative
what’s a semiconservative mode of replication?
New DNA duplex consists of one old
strand (parental) and one new strand
(daughter)
what’s a conservative mode of replication?
New DNA complex consists of two newly
synthesized daughter strands leaving the
parental duplex intact
who discovered that DNA replication is
semiconservative?
Matthew Meselson & Franklin Stahl
How did they discover the semi-conservative model of replication?
Nonradioactive “heavy” nitrogen isotope (15N) to tag
parental DNA strands
DNA molecules with different densities were separated
by a special type of centrifugation
what’s the Meselson & Stahl
Experiment
How are proteins involved in replication?
- DNA polymerase - main molecule synthesizing DNA strands
- Helicase - responsible for unwinding DNA
*
The function of sliding DNA clamp
•Protein that encircles DNA and attaches to the rear of DNA polymerase (relative to
forward movement)
•Tethers DNA polymerase to the template strand and increases the rate of DNA
synthesis
DNA polymerase structure
thumb structure
where does replication starts?
origin of replication (ori)
function of topisomerase
prevents twisting as DNA unwinds
function of single stranded binding proteins SSBs
coat and stabilize single stranded DNA, preventing two strands from re-forming double stranded DNA
what’s okazaki fragments?
DNA polymerase copies the other strand in short lengths
synthesis of lagging strand occurs in small discontinous stretches
what is discontinuous replication?
The synthesis of a new strand of a replicating DNA molecule as a series of short fragments that are subsequently joined together.
what’s leading strand ?
- In DNA replication, the new DNA strand synthesized in the direction of DNA unwinding
- Synthesized on the leading strand template
- One primer is required
•Replication continues to the end.
•The strand is replaced by DNA when the last fragment
catches up to it.
what’s lagging strand?
•New DNA strand synthesized discontinuously, in the direction opposite DNA
unwinding
•Synthesized on the lagging strand template
- Multiple RNA primers are required.
- The final primer is added 100 nucleotides from the 3’ end of
the template leaving the new daughter strand shortened by
about 100 nucleotides.
DNA polymerase ass nucleotides only to an existing strand
T/F
T
How does a new strand beings and what enzyme is used?
- new strand begins with a short chain of RNA primer synthesized by enzyme primase
- Primase leaves the template, and DNA polymerase takes over, extending the RNA
primer with DNA nucleotides as it synthesizes the new DNA chain - RNA primers are replaced with DNA later in replication
How does DNA plymerase correct its own errors?
- proofreading, detects the mispairing of improper hydrogen bonds
- when the error is detected DNA polymerase activates cleavage function removes and replace
what is origin of replication?
- each point where DNA replication is initiated
- The opening of the double helix at each
origin of replication forms a replication
bubble with a replication fork on each side.
large linear chromosomes have one origin of replication
T/F
F many origins of replication
examples of circular DNA
mitochondria, chloroplasts
how do circular DNA replicate?
- there is a single origin of replication
- Replication proceeds in both directions
until the replication forks meet and fuse
on the opposite side, completing one
round of replication.
what is telomere?
End of a eukaryotic chromosome is capped by a repeating sequence called the telomere
How many telomeres do humans have?
In humans, the telomere consists of the
sequence 5’-TTAGGG-3’ repeated
approximately 1500-3000 times.
how many base pairs are lost by chromosomes after replication?
50–200 base pairs
when does the cell die?
after 20-30 divisions
what is telomerase?
enzyme that adds nucleotides to telomeres, especially in cancer cells.
what is PCR?
Polymerase chain reaction - Cyclical process that makes
millions of copies of a section
of DNA
what are the steps of PCR
Each cycle of amplification includes three steps
- Denaturation
- Annealing
- Extension
what contains in the PCR mix?
Genomic DNA (contains target)
Primer A
Primer B
Buffer
dATP
dGTP
dCTP
dTTP
DNA polymerase
Define denaturation in PCR
A solution containing double-stranded DNA (the template duplex) is heated to separate the DNA into two individual strands.
Define annealing in PCR
When the solution is cooled, the two primers anneal to their complementary sequence on the strands of the template duplex.
Define extension in PCR
DNA polymerase synthesizes new DNA strands (complementary to
the template duplex strands) by extending primers in a 5’ to 3’ direction.
At what temperature is DNA polymerase optimal?
37 C
whose DNA polymerase does not denature at high temperature?
Thermus aquaticus (bacterium) – lives in hot springs at extremely hot temperatures
what is PCR used for?
- Author Ricki Lewis (Human Genetics), lists some very interesting applications:
- A preserved quagga (a relative of the zebra) and a marsupial wolf, both extinct.
- Poached moose meat in hamburger.
- The brain of a 7,000-year-old human mummy.
- The digestive tracts of carnivores, to reveal food web interactions.
- Roadkill and carcasses washed ashore, to identify locally threatened species.
- Products illegally made from endangered species.
- Genetically modified bacteria released in field tests, to follow their dispersion.
- One cell of an 8-celled human embryo to detect a disease-causing mutation.
- Remains in Jesse James’s grave, to make a positive identification.
- The intestines of genital crab lice on a rape victim, which matched the DNA of the suspect.
- Fur from Snowball, a cat that linked a murder suspect to a crime.
what is gel electrophoresis ?
In electrophoresis, DNA molecules separate by size. Larger molecules move more slowly because they take longer to work their
way through the pores in the gel.
In addition to amplifying DNA, it can be useful to “cut” DNA at specific sites.
T/F
T
what ahppens when you cut pieces of DNA?
allows pieces from the same or different organisms to be brought together in recombinant DNA technology.
where are restriction enzymes sourced from?
bacteria
what is restriction enzyme ?
A restriction enzyme is a protein isolated from bacteria that cleaves DNA sequences at sequence-specific sites, producing DNA fragments with a known sequence at each end
what is southern blot?
a procedure for identifying specific sequences of DNA, in which fragments separated on a gel are transferred directly to a second medium on which detection by hybridization may be carried out.
what goes in the sequencing PCR mix?
Genomic DNA (contains
target)
Primer A
Primer B
Buffer
dATP
dGTP
dCTP
dTTP
DNA polymerase
ddATP*
ddGTP*
ddCTP*
ddTTP*
what is dna sequencing ?
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA.
what is recombinant DNA
- Recombines DNA from two or more sources
into a single molecule - The donor fragment may be a protein coding
gene, a regulatory part of a gene, or any
DNA segment of interest. - Vector carries the donor fragment (often a
bacterial plasmid.
what is transformation of DNA ?
- The recombinant DNA is introduced into a bacterial cell by means of transformation, in which bacteria are chemically induced into a state in which they take up DNA from outside the cell.
- As the bacterial cell replicates and divides, it also replicates the recombinant DNA.
what is DNA editing
DNA is inserted, deleted, modified or replaced in the genome of a living organism.
