Gene Expression and Laboratory Techniques Flashcards
What is the first step in oncogenesis?
Tumor initiation
involves changes that allow a single cell to proliferate abnormally; cell bypasses regulatory steps of cell cycle that normally prevent proliferation
occurs as a cell develops the ability to proliferate even more aggressively as its descendants are preferentially selected for and come to predominate the growing tumor
tumor progression
malignant cells often unergo mutations allowing them to secrete growth factors to stimulate their own growth
when cancer cells gain the ability to migrate to other parts of the body
metastasis
what enzymes in a cancerous body digests components of the extracellular matrix and favor metastasis?
proteases
promotion of growth of new blood vessels
in cancerous body, these vessels feed the growing tumor
angiogenesis
mutagenic compounds
carcinogens
____ is associated with mutations that occur by random chance, as a result of carcinogens, and/or the dysregulation of gene expression
Name the RNA
what type of RNA activity is abnormal in cancerous cells
miRNA
induces the growth of proliferative cells by stimulating te activity of proteins involved in growth and division
tumor promoters
genes that promote abnormal growth and proliferation
oncogenes
genes that function to prevent tumorigenic properties
tumor suppressor genes
mutation-induced dysfunction -> cell cannot protect itself
main functions: repressing expression of genes that are essential to cell cycle progression, ensuring cell responds appropriately to DNA damage at cell cycle checkpoints, repairing DNA damage, preventing changes in adhesion & proliferation involved metastasis
What are the two main genes involved in oncogenesis?
- oncogenes
- tumor suppressor genes
a cancer-causing virus
tumor virus
e.g Hepatitis B (liver cancer)
many tumor viruses contain retroviral oncogenes, which are reverse-transcribed into the DNA of infected cells. These oncogenes i.e RAS,RAF, and SRC often encode proteins that are key components of signaling pathways that stimulate cell proliferation. Tumor viruses can also hinder the correct function of tumor suppressor genes or promote the function of oncogenes
genes that code for growth factors, receptor tyrosine kinases, transcription factors, and regulatory GTPases. They function as oncogenes after mutation or inapropriately elevated expression
proto-oncogenes
turns into oncogene if it mutates in a way that increases its activity
TP53 is a ____ gene that encodes the p53 protein involved in responding appropriately to cell damage
tumor suppressor
the ____ gene is implicated in half of human cancers
TP53
encodes the p53 protein involved in responding appropriately to cell damage
the ____ genes are responsible for repairing and responding to DNA damage
BRCA
implicated in breast and ovarian cancers, have heridary bases
DNA sequence that upregulates
enhancer
DNA sequence that downregulates
silencer
regulatory proteins
transcription factors
covalent modifications of histones that upregulates gene expression
acetylation
covalent modifications of nucleotides that downregulates gene expression
methylation
non-coding RNA sequences that downregulate gene expression
siRNA, and miRNA
what’s another name for a restriction enzyme
restriction endonuclease
enzymes that cleave DNA at specific recognition sites
restriction endonucleases
4 to 8 base pair sequences that are recognized by a restriction enzyme to cleave DNA. Have symmetry of palindromic sequences
recognition site
when a sequence of nucleotides of complementary strands of DNA read the same in both directions, either from the 5-prime end or the 3-prime end
palindromic sequences
describe blunt vs sticky ends in relation to restriction enzymes
blunt ends: when restriction endonucleeases cleaves a DNA sequnce vertically across the recognition site
sticky ends: when restriction endonecleases cleaves a DNA sequence in a zig-zag fashion
What is the significance of restriction enzymes?
They cut DNA into fragments that can be “tied” back together by DNA ligase w/o necessariy respecting the original location of the sequences (recombination)
DNA molecules used to carry genetic material into a cell where it can be replicated or expressed. Used in DNA recombination processes
vectors
plasmids and bacteriophages
vectors in the form of viruses that infect bacteria. They strip out non-essential genes to carry recombinant sequences
bacteriophages
short circular DNA molecules that can replicate independently in bacteria
plasmids
recombinant plasmids carrying human DNA inseerts can be introudiced into E.coli where they replicate along with the bacteria to yield millions of copies of plasmid DNA. The plasmid DNA can then be isolated, generating recombinant molecules containing a single fragment of human DNA that can be analyzed and further manipulated
Describe the steps in the DNA cloning process
- Host plasmid is cleaved by restriction enzyme
- DNA fragment of interest is inserted into plasmid
- Plasmid is annealed by DNA ligase
- Recombinated DNA is introduced to a bacteria
- Bacteria carrying the plasmid are selected and reproduce
- Bacteria replicate the plasmid and pass it on to their offspring, making copies of the DNA it contains
- DNA/protein is eventually harvested and purified for use
antibiotic genes that can kill of bacteria cells that did not take up plasmids, so only cells with plasmid remain
during DNA cloning/purifying process
antibiotic resistance gene
genes that distinguish betwen recombinant and non-recombinant plasmids
during DNA cloning process
reporter genes
these genes code for a product that creates a obvious phenotypic change (i.e change in color) and contains recognition sites for restriction enzymes that is used in restriction. Thus, E.coli with non-recombinant plasmids will express the reprter gene normally, but those with recombinant plasmids will not, allowing them to be distinguished and subcultured
Name the process
What does this image depict?
DNA recombination via plasmid vector
Explain why a researcher may want to use a bacteriophage instead of a plasmid as a vector
bacteriophages can be used for longer sequences. Plasmids generally contain 2-4kb of DNA, bacteriophages can contain up to 15kb
RNA sequences can be cloned through the generation of ______
complementary DNA (cDNA)
which enzyme can synthesize a DNA copy of RNA?
reverse transcriptase
Descripte the steps in RNA cloning
- synthezize DNA copy of RNA (cDNA) via reverse trancriptase
- cDNA is ligated to vector DNA
an organism whose genome has been modified
transgenic organism
for research purposes
organism in which one or more genes have been disabled
knockout organism
for research purposes
involves splicing a functional allele into the cells of a patient with nonfunctional alleles, devastating genetic disorders
gene therapy
special cells that have the ability to develop into many different cell types, from muscle cells to brain cells
stem cells
a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size and/or charge
gel electrophoresis
note: nucleic acids have a high degree of negative charge, thus they will migrate from cathode and towards anode
note #2: there is an inverse relationship between molecular size and distance migrated
molecular-weight size markers that can be run parallel to the DNA of interest in an electrophoresis experiment to identify the approxiate size of the DNA fragments analyzed
ladders
the ability of a single strand DNA or RNA to join with complementary base pair sequences
hybridization
important for PCR, watch KA video
a specific DNA or RNA fragment that can be labeled radioactively. These are used to detect the presence of nucleotide sequences in analyzed RNA or DNA that are complementary to the sequence in the _____
hybridization probe
technology used to identify specific **DNA ** sequences
southern blotting
technology used to identify specific RNA sequences
northern blotting
p. 118
a laboratory technique used to detect a specific **protein **in a blood or tissue sample.
Western blotting
microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence or gene
DNA microarray
Uses dideoxynucleotides to terminate synthesis ad elecrophoresis to analyze fragment size
a short piece of single-stranded DNA that binds to the template DNA and acts as a “starter” for the polymerase
primer
used to make exponentially large number sof copies of DNA in a short amount of time using primers
polymerase chain reaction (PCR)
What are differences between Sanger sequencing and PCR?
Sanger sequencing is used to generate every possible length of DNA up to the full length of the target DNA while PCR is used to duplicate the entire DNA sequence
