• Shuffle
    Toggle On
    Toggle Off
  • Alphabetize
    Toggle On
    Toggle Off
  • Front First
    Toggle On
    Toggle Off
  • Both Sides
    Toggle On
    Toggle Off
  • Read
    Toggle On
    Toggle Off
Reading...
Front

Card Range To Study

through

image

Play button

image

Play button

image

Progress

1/93

Click to flip

Use LEFT and RIGHT arrow keys to navigate between flashcards;

Use UP and DOWN arrow keys to flip the card;

H to show hint;

A reads text to speech;

93 Cards in this Set

  • Front
  • Back
Gene Expression:


RT-PCR: reverse transcription polymerase chain reaction. There are two steps.
In the first, RNA is reverse transcribed to make cDNA of interest.
In the second, normal PCR is used to amplify the number of copies of that individual cDNA. (remember in RT-PCR we don’t need to denature on the first step because we already have a single strand.)
Production of a protein translated from an mRNA, which was transcribed by a DNA sequence. There are some exceptions to this definition. For example, some RNA’s are functional without need for protein and some cells will begin making a protein but stop halfway through.
cDNA:
Complementary DNA. It is complementary and antiparallel to the RNA strand which produced it.
RT-PCR:
Reverse transcription polymerase chain reaction. There are two steps.

1. RNA is reverse transcribed to make cDNA of interest.
2. Normal PCR is used to amplify the number of copies of that individual cDNA.

(remember in RT-PCR we don’t need to denature on the first step because we already have a single strand.)
How are cDNA's produced?
An oligonucleotide primer (which is a small piece of DNA) is annealed to the mRNA. Reverse transcriptase starts from this primer and sythesizes the cDNA from the template RNA. Then, the mRNA is degraded with alkali leaving only the single strand of cDNA. Then a new primer is annealed and DNA polymerase I extends the complementary sequence of that cDNA.
How are cDNA's useful?
They’re useful because they only contain portions of DNA that are expressed (no introns). They can be cloned.
Q-RT-PCR:
RT-PCR that allows both amplification and detection of a sequence at the same time using a florescent (taqman) probe that is activated when the cDNA is duplicated.
Reverse Transcription:
using an RNA strand as a template and using the reverse transcriptase enzyme to make a cDNA.
Reverse Transcription:
Using an RNA strand as a template and using the reverse transcriptase enzyme to make a cDNA.
Reverse Transcriptase:
Enzyme used in reverse transcription to make cDNA from an RNA template.
Microarray:
Experiment used to measure gene expression at the level of RNA. It is used to determine what genes are expressed in a cell at a given time. It can be used to measure gene expression on a large scale in a genome.
Differentiate between the methods used for PCR, RT-PCR, and Q-RT-PCR:
PCR is for amplifying DNA uses Taq Polymerase, RT-PCR is for amplifying RNA (by creating many copies of cDNA) uses reverse transcriptase to create the cDNA then Taq Polymerase to duplicate the cDNA, Q-RT-PCR amplifies cDNA and simultaneously allows the gene to be detected. Uses Reverse transcriptase Taq Polymerase, and florescent probes.
Northern Blotting:
Cheap, shows RNA splicing, One gene at a time, less sensitive
The methods to quantify and detect gene methods:
Northern Blotting
RNA Splicing
RT-PCR
Q-RT-PCR
Microarray
RT-PCR:
Sensitive, less expensive, challenging, semi-quantitative.
Q-RT-PCR:
Most sensitive, quantitative, expensive, a few genes at a time
Microarray:
info on many genes, sensitive, relative quantification, expensive, no info on splicing
Genetic Polymorphism:
Differences in DNA sequences within a population
Frameshift:
Any deletion or insertion that is not 3 nucleotides in length.
Silent:
A change in the nucleotide sequence that does not change the amino acid.
Substitution:
One of the nucleotides is swapped out for another.
Insertion:
Adds nucleotides to the DNA sequence making the molecule slightly longer. Can have a huge impact if not in multiples of three.
Deletion:
Nucleotides are removed making the DNA molecule slightly shorter. Can have a huge impact if not in multiples of three.
Why may or may not polymorphisms have an impact on the phenotype of an organism?
Not all of DNA codes for proteins, there are a lot of redundant nucleotides and empty space, only a small percentage of DNA actually codes for proteins. Also the mutation might be a silent mutation where the amino acid is not changed. There is also a chance that the function of the protein will not be affected by the change in an amino acid or that there will be minimal loss of function.
Why do frameshift mutations cause dramatic phenotypes?
Changes the whole sequence downstream of the mutation thereby changing most of the amino acids.
Histones
Compact the DNA and control access of genes in the DNA to transcription (gene expression) 1.6-2.0 twists of DNA per histone.
Acetylation
Where you add or remove acetyl groups Histone acetylation changes the overall charge of histones (from highly positive to less positive) DNA becomes less attracted to histones and the packing state of chromatin is loosened.
Nucleosome
DNA+histone
Chromosome
Organized structure of DNA that is condensed before cell division and the DNA is evenly partitioned between the two daughter cells. If it was not condensed before cell division, DNA would break as it was pulled apart
Chromatin
DNA that makes up the contents of the nucleus of the cell

Only found in eukaryotic cells, NOT prokaryotic cells
Acetylated vs. deacetylated histones:
Activated vs. deactivated genes
Antibodies:

Be able to explain how DNA can be compacted (via histones and other interactions
Proteins that are designed to be able to bind to highly specific molecules; target specific amino acid sequences; can use them to detect presence of specific proteins
How can DNA be compacted?
DNA is wrapped around histones and then the histones interact with each other to further coil the DNA. The coil is then wrapped around a protein scaffold to compact it even more.
How can modification of histone proteins lead to changes in chromatin structure and gene expression?
When the histone is acytylated then the overall charge of the histone is dropped and the DNA loosens up which allows the DNA to be read and genes to be expressed.
How does ChIP assay work?
Chromatin Immunoprecipitation. It is used to determine what proteins are present in chromatin at specifc genomic locations. DNA is sonicated to break it up into manageable pieces then protein specific antibodies are attached to the desired protein which cause the proteins to become heavy and are centrifuged and separated from the rest of the DNA, the Antibodies and protein are removed and the DNA is run through PCR to test for the desired sequence.
DNA polymerase I:
Fills in gaps left when the RNA primers are removed and removes the last RNA nucleotide left over from Rnase. Only works with Okazaki fragments. Has proofreading capacity to correct its own mistakes
Topoisomerase:
Creates “nicks” in the DNA strand to relieve the pressure that comes from the DNA getting coiled upstream from the replication fork.
Telomerase:
Extends the ends of newly replicated linear chromosomes to fix the “blunt ends” to preserve the length
Lagging Strand
Due to the orientation of the 5’ and 3’ ends, this strand is synthesized piece by piece and is later ligased together.
Leading Strand:
Due to the orientation of the 5’ and 3’ ends, this strand is synthesized directly with no breaks. Longer than the lagging strand
Dimer
A protein composed of two subunits.
Semi-discontinuous DNA replication:
The leading strand is replicated with little to no breaks while the lagging strand is replicated in segments which are later ligased together.
Semi-Conservative DNA Replication:
One parent strand is hybridized to one daughter strand when the DNA is replicated.
Pulse-chase Experiment
Add a pulse of a labled substance such as radioactively labeled nucleotides or amino acids. These are incorporated in to newly synthesized molecules. Then remove the pulse label and restore regular unlabeled molecules (the chase). Observe what happens to the labeled molecules over time to track them.
Ligase
Seals DNA fragments together
DNA polymerase III:
Synthesizes the new DNA strands. Dimer, one copy does all of the work on the leading strand the other does a lot of the work on the lagging strand. Has proofreading capacity to detect its own mistakes.
RNAse H
Removes the RNA primers except for the last RNA molecule
Primase
Synthesizes small primers of RNA to start the replication process. Creates a 3’ end for DNA polymerase to grab on to.
Helicase
Unwinds the DNA so that it can be replicated.
Goals of a cell when performing DNA Replication
Make complete new copies of genetic material in preparation for cell division so that each daughter cell has same genes/traits as parent cells.
Maintain genetic information with accuracy
Explain why synthesis of the 2 different DNA strands must be fundamentally different from each other
Remember, all DNAs and RNAs are polymerized in the 5’ to 3’ direction (you can only add onto the 3’ OH and not onto a 5’ P)
Initiator contribution to DNA replication:
recognizes ori sequence and bind to them and starts to unwind DNA. Recruits other proteins.
Helicase contribution to DNA replication
Breaks hydrogen bonds to open DNA template(makes 2 single strands from one double strand). Makes DNA into a suitable template molecule.
Single Stranded DNA Binding Proteins contribution to DNA replication
Keeps DNA as a suitable template molecule.
Primase contribution to DNA replication
Synthesize small primers made of RNA to start the process. Provide 3’-OH ends for extension.
DNA Polymerase: Adds nucleotides(pre-existing 3’-OH required). Uses template strand to determine which nucleotides to add.
What are the problems associated with having a linear DNA genome (like the human genome)?
- leading strand is supposed to be longer than lagging strand, but how can it be longer without a template to direct the sequence?
- neither leading/lagging strand can be entirely complete w/o assistance; our linear chromosomes would get shorter w/ every division
Error Rate:
Number of Errors produced by DNA polymerase. 1 in 104 to 105 plus DNA polymerase has proofreading which corrects its own mistakes and mismatch DNA repair corrects another 99-99.9% of mistakes. Permanent errors occur every 1 in 108 to 1011 bp of DNA per diploid cell
Retrovirus:
Viruses that are obligated to integrate into the host genome in order to replicate. The Viral DNA is integrated into the host DNA which can cause the disruptions of genes.
Transposable Element:
Segments of DNA that can be moved around or transposed to different areas of the genome of a cell. Sometimes can be transposed into the middle of a gene and disrupt the function of the gene.
Mismatch
DNA bases don’t line up and hydrogen bonding is disrupted
Alkylation:
Process by which alkyl’s are added to something. In this case it means adding alkyl’s on to DNA bases which can cause them to change to a different base.
Hydrolytic Cleavage:
Water damage. Can break phosphodiester bonds, which can be repaired by ligase. Can break N-glycosyl bonds which leads to depurination (no base) and can also break the bonds linking exocyclic amine groups to bases (bases can potentially change to different bases)
Mutagen
Environmental conditions that can cause DNA damage.
Proof reading
Method by which the DNA polymerase checks its own work, DNA polymerase can fix around 99-99.9% of its own mistakes
Environmental mutagens that can cause DNA damage:
UV radiation (Sunlight)
Ionizing radiation (X-Rays, Gamma Rays)
Hydrolytic cleavage (Water)
Oxidation (free radicals)
Alkylating agents
Chemical cross-linking agents
How a DNA base can be changed to a different base through DNA damage:
Deamination can cause the bases to change to another base. The Methylated cytosine base can get deaminated which would cause it to be changed to a thymine.
Loss-of-function mutation
A mutation that causes a gene to stop working.
Gain-of-function mutation
A mutation that causes a gene to become functional.
How does the Ames test works to identify potential DNA mutagens?
Why this method is superior to monitoring for loss-of-function mutations?
Tests for potential mutagens by using a bacterial strain that cannot produce histidine due to a point mutation. If the mutagen is potent enough it can fix the point mutation and allow the bacteria to form colonies.
The 3 main mechanisms by which DNA damage is detected:
DNA repair enzymes which scan the DNA surface for bulges, kinks or holes
DNA replication
RNA transcription enzymes which discover the damage when they use DNA as a template
Differentiate between the types of mutations repaired, and the mechanisms used, in the 3 main types of DNA repair
Direct Repair: Nothing removed or replaced, the damage is simply reversed, used for minor damage. Photoreactivating enzyme can use light to reverse thymine dimers. O6-methylguanine Methyl Transferase (MGMT) (sweet band BTW! :D) selflessly gives its life to remove methyl groups and repair the damage

Excision Repair: Two types
Base excision repair: Can fix single damaged bases due to: Depurination, Deamination, Oxidation, and Alkylation.
Nucleotide excision repair: Can fix Pyrimidine dimers. Multiple nucleotides are removed, and DNA Polymerase I fills in the gap and then the two ends are ligated together.

Mismatch Repair: Base pairing mismatches, short insertions/deletions following DNA replication are repaired using similar mechanisms to excision repair.
Homologous sequences:
2 DNA molecules with very similar DNA sequences in the same order
DNA recombination:
Process of introducing new DNA sequences into existing DNA (often includes swapping sequences between 2 strands of dsDNA)
Crossing over:
homologous DNA sequences from different chromosomes line up next to each other and switch places
Genetic linkage:
A method to determine how close 2 genes are to each other based upon the frequency of crossing over together vs. crossing over separately
Holliday junction:
A mobile junction of four strands of DNA
Knockout Mouse:
A mouse strain that is genetically engineered to be lacking a specific gene
Neomycin:
Gene that provides resistance to a specific drug to help the cell survive
Chimera:
A single organism (usually an animal) that is composed of two or more different populations of genetically distinct cells that originated from different zygotes involved in sexual reproduction.
Selectable marker
A gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory to indicate the success of a transfection or other procedure meant to introduce foreign DNA into a cell.
Heterozygote
both alleles are different
Homozygote:
both alleles are the same
Knockin Mice:
A mouse strain that is genetically engineered to have a new gene that is normally lacking.
Conditional Knockout:
Allows us to grow a mouse up to a certain developmental stage and then inactivate the gene of interest in a particular tissue to see what goes wrong. The gene remains functional during development and is then deleted out of specific types of cells later on
Genetic linkage:
A method to determine how close 2 genes are to each other based upon the frequency of crossing over together vs. crossing over separately
Holliday junction:
A mobile junction of four strands of DNA
Knockout Mouse:
A mouse strain that is genetically engineered to be lacking a specific gene
Neomycin:
Gene that provides resistance to a specific drug to help the cell survive
Chimera:
A single organism (usually an animal) that is composed of two or more different populations of genetically distinct cells that originated from different zygotes involved in sexual reproduction.
Selectable marker
A gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory to indicate the success of a transfection or other procedure meant to introduce foreign DNA into a cell.
Heterozygote
both alleles are different
Homozygote:
both alleles are the same
Knockin Mice:
A mouse strain that is genetically engineered to have a new gene that is normally lacking.
Conditional Knockout:
Allows us to grow a mouse up to a certain developmental stage and then inactivate the gene of interest in a particular tissue to see what goes wrong. The gene remains functional during development and is then deleted out of specific types of cells later on