DNA Replication Flashcards
What is the central dogma?
DNA to DNA Replication
DNA to RNA Transcription
RNA to Protein Translation
What is reverse transcriptase?
It is a exception to the central dogma where the RNA genome of retroviruses is converted to DNA. This DNA then is used to make new RNA molecules by the virus infiltrating the host genome.
RNA to DNA to RNA
What is an example of a _________ that uses reverse transcriptase?
Retrovirus
HIV
What is RNA dependent RNA polymerase?
RNA genome of RNA virus is copied to new molecules of RNA
What is an example of a _____ that utilizes RNA dependent RNA polymerases?
virus
COVID
Nucleoside is made of :
Nucleotide is made of:
purine/pyrimidine nitrogenous base and pentose ribose/deoxyribose sugar
Nucleoside + phosphates
DNA is a polymer of _____ (#) ___________.
The purines are :
The pyrimidines are:
4 deoxyribonucleotides
Adenine, Guanine
Cytosine, Thymine
RNA is a polymer of _____ (#) ___________.
The purines are :
The pyrimidines are:
4 ribonucleotides
Adenine, Guanine
Cytosine, Uracil
Purines are made of _______ ring(s) and include ____ and ___.
Which one has only one NH2 group ?
2 rings
Adenine and Guanine
Adenine
Rings as PURE As Gold
Rings - 2 Rings
Pure- Purine
As Gold - Adenine and Guanine
Purines are made of _______ ring(s) and include ____ and ___.
Which one is associated with RNA?
1 rings
Thymine, Uracil, Cytosine
Uracil
What are the precursors for DNA or RNA synthesis?
Triphosphates (dNTPs or NTP)
____________ are incorporated into DNA or RNA and ______ is released.
Monophosphates
PPi (pyrophosphate)
DNA and RNA is formed by linking ____ by ________. It grows in the ___ to ___ direction.
Describe it.
Nucleotides , Phosphodiester bond
5’ to 3’
The 5’ phosphate group on one nucleotide is joined to the 3’ hydroxyl group of the pentose sugar of the prior nucleotide.
This releases PPi
RNA is hydrolyzed rapidly under ___________ conditions because of the __________ on the _________. DNA is _____ in such conditions.
alkaline
2’ OH on the pentose sugar
stable
On DNA, the 2’ carbon on the _______ contains a ______ while on RNA a ______ is present.
pentose sugar
H, OH
1’ carbon pentose sugar
2’ carbon pentose sugar
3’ carbon pentose sugar
4’ carbon pentose sugar
5’ carbon pentose sugar
1.Holds the purine or pyrimidines
2: H or OH to distinguish RNA vs DNA
3: Participates in phosphodiester bond linkage as nucleotide be added TO. (OH)
4: Provides molecules stability and hosts 5’ carbon
5: Participates in phosphodiester bond linkage as adding nucleotide by using phosphate group.
Adenine and _______ form _______ bonds
Guanine and _______ form ______ bonds
Adenine and Thymine form two hydrogen bonds
Guanine and Cytosine form three hydrogen bonds.
GC3 rhymes hehe
The DNA structure is ___________ wound around each other to form ___________. Each “turn” is made of __________.
Coiling of the DNA creates ____________.
two sugar phosphate backbones wound around each other to form a right-hand double helix.
Each turn is made of 10.4 nucleotides.
Major groove and minor groove
DNA replication is _______________. This means that the parental DNA double helix will __________.
Semi-conserved and discontinuous
serve as template strands for two new complementary DNA strands.
Each new replicated piece has one old and and one new piece of DNA.
DNA must be ____ and both the _________ and ________ copied using ________.
DNA can only synthesize in _______.
unwound
5’-3’ and 3’-5’
DNA polymerase
5’-3’
_______ is the assembly of enzymes that replicate DNA. These enzymes are______.
In bacteria ________ proteins are involved in the _____.
Repliosome
Helicase
DNA Polymerase
DNA primase.
20-25 , repliosome
ALL DNA polymerase _________________.
Synthesize 5’-3’
Require a template
Require a primer with a 3’ OH
Use dNTPs as energy source
Incorporate dNMPS into DNA
The ________ strand is continuous while the _______ is not.
Leading, Lagging
Why is the lagging strand replication of DNA discontinuous?
How is the discontinuity addressed?
The lagging strand is the “5’-3’” strand of DNA. When the replication fork opens up in the 5’-3’ direction, DNA polymerase will run into the 5’ end of the DNA strand if it proceeds to add nucleotides in a 5’-3’ fashion (aka the reverse direction of the OG 5’-3’ DNA strand).
Instead, DNA is synthesized in small fragments called Okazaki fragments that are eventually synthesized together to create one long lagging strand.
DNA Replication Steps
- _________ unwinds DNA ahead of the _______ and __________ straightens DNA template and _________.
- The ______ is synthesized in the 5’-3’ direction by _________. Only one _____ is needed at the start of replication for the ________.
- ______ are synthesized on the ________ by ________.
- ____ are elongated by _______ in the lagging strand. ______ are formed.
- _______ is removed by a _______.
- Another molecule of ________ fills the gaps between Okazaki fragments where the _________ was but still leaves a _______.
- ______ joined the nick between Okazaki fragments.
- ____ prevents ______.
- DNA helicase unwinds DNA double helix ahead of the replication fork
SSB proteins (Single Strand Binding) straightens out DNA template and facilities DNA polymerase
- leading strand , DNA polymerase , RNA primer, leading strand
- RNA primers are synthesized on the lagging strand by DNA primase.
- RNA primers, DNA polymerase. Okazaki fragments
- RNA primer is removed by a 5’-3’ exonuclease (RNAse H)
- DNA polymerase, RNA primer, nick
- DNA ligase
- DNA topoisomerase prevents DNA tangling
What is DNA Primase?
It does NOT require _____ and can join ________.
DNA primase creates short RNA molecules complementary to the DNA template in the 5’-3’ direction in intervals of about 100-200 nucleotides along the lagging strand.
It does NOT require a primer and can join two nucleoside triphosphates.
RNAseH is responsible for _________.
Exonuclease
Removing RNA primer from lagging strand.
DNA topoisomerase is responsible for __________.
preventing the tangling of DNA
What is the origin of replication?
The position in the nucleotide sequence where the DNA helix is opened. A replication bubble is formed.
Prokaryotic organisms have ___________ origin of replication while eukaryotic organisms have ______.
1 / many
Why do eukaryotic organisms have so many origins of replication?
Eukaryotic organisms have multiple origins of replication to allow for the synthesis of the complete genome in a reasonable amount of time
Replication origins are activated in clusters called ____________.
Replication units are activated at different times during the ________of the cell cycle. Within one replication unit origins are spaced ________ based pairs apart.
replication units
S-phase
30K-250K
If you periodic cells have multiple origins of replication, how does the replication
Individual replication forks proceed from each origin stopping when they collide with a replication fork from the opposite direction.
List the following processes from most prone to error to least.
5’-3’ polymerization
3’-5’ exonucleolytic proofreading
+Strand-directed mismatch repair
Analysis of DNA can be done by_________
Sequencing
PCR/qpcr
SNP arrays
Genome Sequencing
What can analysis of RNA be done by and how?
Reverse transcriptase-PCR / RT-qpcr.
In RNA the expression of a particular gene is used to analyze specific molecules.
RNA-seq
What can analysis of proteins be done by and how?
Western Blotting and Immunohistochemistry
Is the protein expressed?
Proteomic
Describe Sanger Sequencing
Sing DNA sequencing, involves en somatic DNA synthesis, using base specific chain terminators and a radio labeled primer.
The base specific chain terminators will terminate at a at C at G and T in four separate rounds.
The DNA products are then denatured on large poly acrylamide gels that can resolve sizes of these products by one nucleotide this will give us different sizes of products ordered from smallest at the end of the polyacrylamide gel to largest at the top, by which we can order the bases of the nucleotides.
Sanger sequencing determines nucleotide sequence in a linear piece of DNA.
What are Dideoxy bases?
Dideoxy bases are kind of altered nucleotide used in Sanger sequencing
They do not contain a hydroxyl group on the three prime carbon of the pentose sugar which terminates the DNA sequence synthesis.
Automated DNA sequencing with fluorescent primers
The concept is similar to Sang sequencing, except the four reactions of each nucleotide contain the same primer with a different fluorescent label. Reactions are then combined at once prior to separation, and the signal is detected by a laser.
Gels not needed!
PCR
What is the product?
PCR is a cycle of heating and cooling DNA strands to replicate a specific region of DNA using an upstream and downstream primer (for the two strands)
The PCR product is the size of the primers and base pairs in between.
When is the defined PCR product made?
Cycle 2
Since PCR involves high temperatures, what molecule is involved in the process that must withstand it?
DNA polymerase developed from bacteria in hot springs in Yellowstone
Restriction Fragment Length Polymorphisms RFLP analysis
If a mutation of a DNA changes the restriction site, you can PCR the region around the possible mutation and subject it to a restriction enzyme.
If the predicted fragment occurs, the mutation is present which can be a diagnostic tool
Applications for Southern Blotting
Variable Number Tandem Repeat Analysis
Many areas of the genome vary in number from person to person. There are thousands of these regions that vary in number that is unique to each individual.
Tells you IF sequence is present and how much
DNA sequencing tells you what the sequence is.
Applications in Medicine of RFLP and VNTR include
Looking for mutations
Embryo typing and genetic counseling for Huntingtons disease
Invitro fertilization
Remission test in cancer patients
Virus Detection
qPCR
What is a key feature that helps qPCR work?
Measures the amount of PCR product after each reaction
A third primer is used that is complementary to sequences being amplified by the two primers. It contained flourophore and is bound by a quencher when that sequence is not present.
When wanted PCR is product is generated, the third probe can attach it self to the PCR product as the third primer should be complementary to the product. This releases the quencher and allows flourophor to activate.
qRT-PCR
Measures RNA levels of specific transcripts (expression of RNA) giving idea of expression changes in disease or experimental conditions.
Western Blotting
Western Blotting separates proteins by weight on a gel. It is transferred to a nitrocellulose membrane and incubated with an antibody that recognizes a specific proteins.
Various assays can be used to detect the antibody and its levels.
Not used for medical or diagnostic purposes!
Immunohistochemistry
Asses protein abundance in cell or tissue including diseased tissue.
Requires LITTLE product
Used for medical and diagnostic purposes
SNP
Single nucleotide polymorphisms
They cause phenotypic differences between people including resistance to human disease or sensitivity to a disease.
SNPs can be used to map disease loci if compared across several samples of the same disease
Genomic DNA is cut with restriction enzyme and amplified using one pair of PCR primers after being ligated into separate fragments. Products are labeled flourescently and hybridized to a microarray with oligonucleotides of each polymorphism to tag them.
RNA-Seq
Utilizes SNP process but at a TRANSCRIBED level of SNP.
Can be used to identify heterogeneous cell populations.
Mass Spectometry
Determine relative protein expression profiles of proteins in and out of cells as well as differences in protein modification.
Uses AA sequence!
Requires high quantity of material.
