Theme 4: DNA Replication and Mitosis - Module 3: Applications of DNA Replication Flashcards
what technique did Kary Mullis develop?
the ability to amplify DNA in a rube vie polymerase chain reaction (PCR) technique
what did this technique allow scientists to do?
to be able to copy (amplify) millions of copies of DNA from very small starting samples
DNA in a tube has revolutionized the world of cellular and molecular biology in what ways?
- has shed light on diagnosis of genetic defeats
- detection of viral DNA in cells
- producing large amounts of DNA from fossils containing trace amounts of DNA
- being able to link specific individuals to DNA samples during forensic investigations
how is a PCR set up?
a sample of DNA is placed into a tube containing a buffered solution with essential ions and salts, along with a pair of short single-stranded DNA primers (usually 15-30 nucleotides in length)
what do the primers do?
bind in a complementary manner to specific regions of the template DNA and serve as starting points for DNA copying
since this is a cell-free system what else is also added within the tube?
free deoxyribonucleotides (dNTPs)
when are the dNTPs utilized?
during the replication process
what does PCR not require?
the multiple enzymes that cells utilize to unwind and stabilize the DNA double helix
where are the tubes placed instead?
in a thermocycler machine
what happens with the thermocycler machine?
it goes through various phases of heating and cooling in automatic programmed steps to facilitate the DNA replication process over various repeated cycles
how many key DNA replication enzymes are still required?
one
what is it?
a special DNA polymerase
what is special about this DNA polymerase?
it is tolerant to high temperatures
why is it added to the tube?
to catalyze the polymerization of each daughter strand within the tube with each replication cycle
what is an example of this type of heat-tolerant DNA polymerase?
Taq polymerase
where was this first isolated from?
the bacterial species Thermus aquaticus - adapted to live in hot springs with temperatures as high as 95 degrees celsius
how many key stages are involved in a PCR reaction
three
what are they
denaturation, annealing, and extension
what does this three step cycle bring?
a chain reaction that produces an exponentially growing population of identical DNA molecules
the types of DNA molecules were referring to depends on what?
the types of primers that are designed
most researchers have a sequence of a gene or DNA segment that they wish to replicate or cone, thus how will researchers design primers?
design primers to bind to or anneal to their complementary sequence on either side of the DNA sequence of interest on the DNA template strands
how must the DNA double helix be at the start of a PCR?
unwound
how is this facilitated?
by a high temperature stage of the reaction
what happens during the denaturation stage?
the reaction mixture is heated to separate the strands of the double-stranded DNA
what will then happen with the thermocycler?
it will cool the solution
what does this allow?
allows the two primers to anneal to their complementary sequences on the DNA template strands on either side of the DNA sequence of interest during the annealing phase
where will the primers bind?
on opposite strands at each end of the target sequence
when does the heat-stable DNA polymerase extend and polymerize the daughter strands?
during the extension phase
what is used?
four dNTPs, starting from the primers and extending the daughter strand in a 5’ to 3’ direction
what does each complete cycle result in?
two double stranded helices containing the desired target sequence portion of the original template DNA
summarize the denaturation step:
a solution containing double-stranded DNA (the template duplex) is heated to separate the DNA into two individual strands
summarize the annealing step:
when the solution is cooled, the two primers anneal to their complementary sequence on the DNA template strands
summarize Extension:
DNA polymerase synthesizes new DNA strands (complementary to the template) by extending primers in a 5’ to 3’ direction
are PCR reactions fast and specific for the DNA sequence that is replicated
yes
with each successive cycle the number of replicated DNA molecules with the same sequence as the parent template duplex is what?
double
what is present after the first cycle of PCR?
two copies of the template duplex - each consisting of one new and one old DNA strand
with each cycle what do the newly synthesized DNA segments serve as?
templates in later cycles
after the second PCR cycle how many copies would be present?
4
what equation can be used to represent the number of copies of the template DNA target sequence
2^n (n=cycles)
what is essential for many of the applications of PCR?
the huge amplification
do the tubes look different after removing them from the thermocycler?
no
what will be in the tube if the PCR was successful?
millions of molecules of amplified segments of DNA
what do researchers use in order to visualize DNA molecules?
gel electrophoresis
what does gel electrophoresis do?
general technique used to separate DNA fragments from other sources, not just from PCR
what else can gel electrophoresis be used to separate?
other macromolecules including RNA and proteins, all on the basis of their rate of movement through an agarose gel in an electric field
where are molecules loaded during DNA gel electrophoresis?
into wells of a porous gel
where do the molecules travel? how?
- along the length of the gel
- because of an electrical field that is applied along the length of the gel
what are the different charges established and where are they located?
positive at one end of the gel and negative at the other end of the gel
what is the charge on DNA and RNA? Why?
- negatively charged
- due to ionized phosphate groups along the phosphodiester backbone
where will they be attracted towards?
attracted towards the positively charged (anode) end of the gel
what also affects how far through the gel the molecules will travel?
size of the molecule
the molecules that travel through the gel at the highest speed tend to be what size?
the smallest molecules
what is the travelling speed and distance of larger molecules?
- slower speed
- smaller distanced along the gel compared to smaller molecules
what is the range of nucleotides that gel electrophoresis can separate?
several hundreds of nucleotides to over 10, 000 nucleotides
what are the PCR results typically?
the amplification of just a single size of DNA molecule
what is it also possible to utilize gel electrophoresis for?
to separate and visualize a DNA sample containing a mixture of DNA fragments of different sizes
what is also loaded onto the gel electrophoresis along with the DNA samples?
a standardized ladder
what is a standardized ladder?
contains DNA fragments of known sizes
what is possible after the actual electrophoresis process?
can visualize the separated DNA fragments
what is used to visualize the separated DNA fragments?
special dyes that intercalate with and stain the DNA fragments
what do these dyes do when exposed to ultraviolet light?
fluoresce
how can the different bands containing the DNA fragments of different sizes be visualized ?
visualization can be detected on the gel using UV light
how is it possible to learn a lot about the function of a gene?
by identifying its nucleotide sequence
along with identifying the nucleotide sequences of important regions that code for functional proteins in our DNA, what can we also identify?
the nucleotide sequences of non-coding regions
why was DNA sequencing developed by Frederick Sanger?
to be able to determine the sequence of a DNA molecule
how was he able to develop this technique?
based largely on his knowledge of DNA replication
what was a limitation to Sanger’s DNA sequencing technique?
it could only determine the sequence of small fragments of DNA
what did Gene Myers and Jim Weber do?
came up with an approach that would revolutionize large-scale sequencing projects and would facilitate the identification of the DNA sequence of the entire genome of any organism
what was this technique refereed to as?
shotgun sequencing
what was this technique based on?
being able to break the entire genome into different sized pieces and then proceed with 3 specific phases
what was the first phase?
Random sequencing of the DNA in each fragment
what was the second phase?
Identifying the regions of overlap between the generated fragments and inferring or assembling the long, continuous sequence of nucleotides in the DNA molecule that makes up each chromosome
what was the third phase?
annotating the sequences to best identify the regions of genomic DNA that encode genes, regulatory regions and even non-coding regions of the DNA
what was critical to the process of whole-genome sequencing?
the development of computational software capable of facilitating the assembly of fragments
when was the whole human genome sequenced?
2000
summarize the three phases
1) sequence DNA
2) assemble sequences
3) annotate the sequences
how is DNA sequencing carried out today?
by sequencing machines
what did early approaches to DNA sequencing use?
the Sanger sequencing technique
what is this technique also known as?
dideoxy chain-termniation method or dideoxy sequencing
since DNA sequencing is based upon our knowledge of DNA replication, during Sanger sequencing, the DNA to be sequenced serves as what?
a template for DNA synthesis
what are the key components required for dideoxy sequencing?
include all the components required for DNA replication
what do these components include?
- denatured, single-stranded template DNA
- short single-stranded DNA primers
- sufficient free deoxyribonucleotides(dNTPs)
- DNA polymerase
what do short single-stranded DNA primers do?
will bind in a complementary manner to specific regions of the template DNA and serve as starting points for DNA copying
how does DNA polymerase link adjacent deoxynucleotides?
catalyzing a covalent bond between the 5’ -phosphate on one nucleotide and the 3’ - OH group on the previous nucleotide
because of this how this occurs the basis of the dideoxy chain-termination method requires what?
the use of modified deoxyribonucleotides
which didexoynucleotides (ddNTPs) will not allow for further elongation of a growing DNA strand?
dideoxynucleotides that are missing the -OH group at the 3’ position
why is this?
the -OH group is required for the attachment of the next nucleotide
as a result of this what did Sanger add into the sequencing reaction tubes in addition to all other components required for DNA replication?
labelled ddNTPs
what will the dideoxynucleotides within the tubes will lead to what?
to a series of interrupted daughter strands
where is each terminating DNA replication located during this?
at the site where dideoxynucleotide is incorporated
during dideoxy sequencing, the sequencing of each daughter strand begins where? and continues until when?
- begins at the 3’ end of the primer
- continues until a dideoxynucleotide is inserted
what happens once a ddNTP is inserted in a daughter strand?
prevents further elongation of the strand
how many molecules of the template DNA does the sequencing reaction contain?
millions of molecules of template DNA
what kind of process is the insertion of the ddNTPs or dNTPs?
random process
between ddNTPs and dNTPs which is added in a smaller amount?
smaller amount of ddNTPs are added relative to the amount of dNTPs
what does this allow for?
the generation of many fragments of many different sizes potentially terminating at every possible nucleotide within a given region
for DNA sequencing to be of us what is it essential to be able to identify?
the molecules in which chain termination has occurred
what must be done to achieve this?
each of the four dideoxynucleotides can be labelled with a fluorescent dye
is the fluorescent dye labelling each of the four ddNTPs the same or different?
different
in this manner what is it possible to do?
distinguish all the chain terminators that are present in all the replicated DNA fragments within a DNA sequencing reaction
after DNA synthesis and chain termination where are the labelled strands in the mixture loaded?
onto a gel and the fragments are separated by gel electrophoresis
how long does electrophoresis continue for?
until each DNA band emerges from the bottom of a gel and a laser excites the fluorescent dye attached to each dideoxynucleotide
what can record the amount of fluorescence that is emitted?
a fluorescent detector
what does the fluorescent detector do after recording the amount of fluorescence emitted?
match this to one of four wavelengths that corresponds to the four fluorescent tags that are attached to each of the ddNTPs
what does the fluorescence detector then do?
distinguishes strands differing in length by as little as one nucleotide
what can then be generated by the laser and detector with every peak corresponding to the nucleotides that make up the DNA sequence that is complementary to the template strand, and always begins after the primer?
a spectrogram trace
what is a limitation of the Sanger method?
it can only determine the sequence of fragments of DNA up to several hundred nucleotides in length
thus what is this technique convenient for?
sequencing short sequences of DNA
what should be used for large stretches of DNA?
shot gun sequencing
what does this technique allow?
the information from multiple DNA sequences to be assembled by examining the regions of overlap between sequenced random DNA fragments
when assembling the sequence of a series of sequenced DNA fragments what occurs?
- the short sequences are examined
- regions of overlap are identified
- short sequences are put together to generate long continuous sequences of nucleotides
therefore what is possible?
it is possible to attain long sequences of large portions of chromosomes on the order of millions of nucleotides in length
what is the assembly of these fragments into one continuous sequence accomplished by?
complex algorithms in an automated fashion through various computer programs
what are contigs?
refers to overlapping DNA segments that are assembled into a consensus region of DNA
what is the assembly of he final DNA sequence based on?
the overlap of sequence similarities between various DNA fragments
consider the sequences of DNA fragments to be represented by sentence fragments of this/a statement. Based on the information in the fragments and identifying the regions of overlap (contigs) between fragments, the full sentence can be assembled in the correct order, generating the complete statement
.
therefore how can the sequences of entire genomes be determined?
based on the sequence similarities within overlapping sequenced DNA fragments
what are researchers able to do once the sequences are assembled?
able to annotate the sequence and identify specific regions of interest along the sequenced DNA
what does annotation of DNA sequences allow?
allows us to obtain a better understudying of the series of A, C, G and T nucleotides that encode all our genetic information in our DNA
is all our DNA in our genome transcribed into RNA? is all our RNA translated into a functional protein product?
no and no
how many possible reading frame are there for any double-stranded DNA sequence?
6
what is one of the first steps following sequence assembly?
to establish the correct reading frame
what are computer programs able to do?
scan the sequence of a genome in both directions and identify each reading frame that is possible on both DNA strands
what is a long stretch of codons that lacks a stop codon identified as?
a good reading frame and indication that that may be the coding sequence
in the example of the human beta glob in gene, only one reading frame will give the full length protein, why is this?
because other reading frame will terminate translation after only a few amino acids
what are annotation softwares able to do as a result?
can identify any gene-sized protein coding stretches of DNA sequences (one reading frames) that lack internal stop codons, but that are flanked on either side by a start and stop codon
what will the computer programs also look for?
typical sequences that code for promoters, or other regulatory sites
are prokaryotic or eukaryotic genomes easily scanned to identify regions of interest?
prokaryotic
why are there different techniques used for eukaryotic genomes?
to identify intron and exon regions amid other regions of the genome
along with identifying the reading farm of a specific DNA sequence, genome annotation requires the identification of?
various patterns (sequence motifs) in the sequenced DNA molecule
protein-coding regions of DNA can be inferred based on?
the identifcaiont of open-reading frames in the DNA or RNA sequence and it consist of triplets of nucleotides that can specify amino acids that will make up the protein and contain no interrupting stop codons
what other sequence motifs can be identified in sequenced DNA?
binding sites for the transcription factors that regulate gene expression
where are these transcription factors located?
upstream, downstream or within introns of a gene
how can some sequence motifs be identified?
from the hypothetical RNA molecule that is inferred from the sequenced DNA molecule
RNA sequences that makes up a transfer RNA (tRNA) molecules forms what?
characteristic hairpin structures - molecule folds back on itself and undergoes complementary base pairing within the molecule itself
how can these sequences be inferred from a DNA molecule?
by looking for nearby complementary sequences within the sequence
do exons and regulatory elements that code for proteins make up a large or small portion of the genome?
small
what do most of the prokaryotic genomes consist of?
genes
what does 50% of the average eukaryotic genome consist of?
repeated sequences that do not code for functional gene products
what were these non-coding repeated sequences originally believed to be?
unimportant/”junk DNA” although may have been found to have their own functions
are there regions in the eukaryotic genome that encodes noncoding RNA as well as many types of repeated, noncoding sequences?
yes
how do these sequences compare in different individuals?
vary from individual to individual and especially across species
what does this contribute to?
the diversity that we can observe across different organisms
