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88 Cards in this Set

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what causes sickle cell anemia?
single nucleotide polymorphism (SNP) in codon 6 of beta-globin gene
what is a haplotype?
combination of alleles at multiple loci that are transmitted together on the same chromosome
what are the two types of gel commonly used for gel electrophoresis?
polyacrilimide
agarose

concentration of either in solution determines size of pores (size of molecules that can be filtered)
which of the macromolecules is larger?
RNA and DNA are much larger than proteins
what is gel electrophoresis?
separation of macromolecules in an electric field through a semisolid matrix
how does blotting work with DNA and RNA?
a filter is placed on top of the gel and dry paper towels are placed on top of that

capillary action draws water into the paper towels and DNA/RNA goes with the water
how does blotting work with proteins?
proteins must be driven out of a gel and onto a filter by an electric field
what is isoelectric focusing?
separating proteins by their essentially unique electric charge at a given pH
what is an isoelectric point?
point at which protein no longer migrates in an electric field because its net charge is zero

occurs when pH = pI
how do DNA and RNA migrate in an electric field?
migrate away from the negative electrode, towards the positive electrode

phosphate backbone gives DNA and RNA a highly negative charge
what is agarose?
carbohydrate extract from seaweed, used for RNA and DNA separation
what type of gel is used for DNA sequencing?
polyacrilamide gel
what happens when proteins are mixed with SDS?
SDS is a detergent, so it interacts with the organic molecules, but it does so in a stoichiometric way (2:1)

since SDS has a highly negative charge, it overwhelms any charge on the proteins it has emulsified and gives all proteins a uniformly negative charge

this allows a person to separate proteins based on size alone
how is a radioactive labelling experiment performed?
whole cells are radioactively labelled and lysed, lysates are then electrophoretically separated and exposed to x-ray film (radioactivity makes a black band)
how is a protein stain experiment performed?
lyse cells, electrophoretically separate lysates, and then stain with something like carboxy blue stain
what happens in blotting if a protein is labelled with an enzyme which produces a colorimetric ppt?
the precipitate stays on the membrane (is visible where proteins are present)
what is used to establish a pH gradient across an electric field?
ampholytes
what are the features of a 2-D gel electrophoresis?
see spots instead of bands
when bands are seen, it indicates one protein which can be present in multiple charge states

proteins are distinguished by both size and charge (very high resolution)
what could give a single protein multiple charge states?
having a variable number of phosphates, etc.
what is a western blot?
target - protein

probe - antibody
what is a northern blot?
target - RNA

probe - complementary RNA or DNA sequence
what is a southern blot?
target - DNA

probe - complementary RNA or DNA sequence
what is a southwestern blot?
DNA on a blot, probed with a protein, which is then probed with an antibody
what is a northwestern blot?
RNA on a blot, probed with a protein, which is then probed with an antibody
how can DNA annealing be removed?
altering salt concentrations
what is the EcoR1 recognition sequence?
GAATTC
what type of end is left by EcoR1?
five-prime overhang
what types of ends can be left by restriction endonucleases?
blunt ends
three-prime overhang
five-prime overhang
what is a common theme among useful restriction endonucleases?
recognize a pallindrome
how many bases are required to anneal pieces of DNA together specifically?
four bases
what three things are required for a chromosome to be considered a chromosome?
telomeres
centromere
origins of replication
what is a plasmid?
circular, autonomously replicating DNA, which is easy to manipulate and makes identification of recombinant molecules easy

unfortunately doesn't carry much DNA (5000-6000bp)
what is bacteriophage lambda?
a large virus which infects bacteria

carries 25000 to 30000 base pairs of DNA
what are YACs?
yeast artificial chromosomes
what are BACs?
bacterial artificial chromosomes
what are restriction endonucleases?
specific class of endonucleases (cut DNA) which cut DNA only at a specific sequence
what is the function of DNA ligase?
to fix nicks in DNA
what is cDNA?
complementary DNA

DNA synthesized from a mature mRNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase
what is the advantage of cDNA (over DNA)?
there are no introns included in the cDNA, so there is much less to sequence
what is required for ALL DNA polymerases?
ALL DNA polymerases require a primer with a free 3' hydroxyl group
what is the primer for the first round of reverse transcription?
complementary sequence (TTTTTTTTTTTTTTT) to poly-A-tail at the 3' end of the RNA
what is the primer for subsequent rounds of reverse transcription?
nicks are made in first strand of RNA and at each nick is found a free 3' hydroxyl which can be used as primers
what is reverse transcriptase?
RNA-dependent DNA polymerase

enzyme encoded by retroviruses which recognizes RNA and makes DNA from it
how can a gene of interest be completely sequenced from genomic DNA?
partially digest the genomic DNA, and use parts of the genomic DNA to "walk" across the gene
what is the advantage of partial digestion over full digestion?
partial digestion provides a range of fragments which overlap with one another, spanning the entire gene
for what reasons would one want to know the sequence of DNA?
structure of gene could give exon/intron boundaries

functional domains of resultant protein
what is sanger ddNTP chain termination sequencing?
samples of target DNA are supplied with DNA polymerase, one dideoxynucleotide (of varying concentration) and all four deoxynucleotides; the result is that DNA will be synthesized and whenever a dideoxynucleotide is inserted the process will stop
what happens as the concentration of dideoxynucleotides is varied in a Sanger ddNTP chain termination sequencing?
the more dideoxynucleotide is in the sample, the more frequently it will be inserted and the more frequently the chains will terminate replication early
how is sanger ddNTP chain termination sequencing performed with modern technology?
use very long, very small polyacrilimide tube with laser at the bottom which stimulates DNA nucleotides (marked with different colors for each) as they are electrophoresed out of the gel; as the last nucleotide put on each time fluoresces, the sequence can be put together
what is the process of PCR?
raise temp to denature DNA
add primer in vast molar excess
lower temp to optimal annealing temp
raise temp to optimal replicating temp
repeat
what is a RFLP?
restriction fragment length polymorphism

a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites
how is RFLP analysis performed?
DNA sample is digested by restriction endonucleases and the resulting fragments are separated according to their lengths by gel electrophoresis

since restriction endonucleases are very specific, even a single base pair difference in the endonuclease site will change the size of the DNA fragments
hos is RFLP analysis performed with PCR?
know a sequence around the RFLP, design primers which span the RFLP, amplify the DNA across the RFLP, digest PCR-amplified DNA with restriction endonuclease and analyze digestion products
what are VNTRs?
variable number tandem repeats

short sequences of DNA which are repeated several times; the sequence is pretty well conserved between people, however the number of times the sequence repeats varies between people
what is ASO?
allele specific oligonucleotide

short piece of synthetic DNA complementary to the sequence of a variable target DNA, which can be used to detect the presence or absence of that certain sequence (so accurately that 1 bp is enough to tell a difference)
how is ASO used to detect DNA differences?
at the right temp and salt concentration, complementary oligonucleotides with a mismatch of just one base will not anneal to one another

a complementary strand is used to probe the DNA present on a blot; if it anneals, there will be a spot (heterozygous or homozygous), but if it doesn't, there will be no spot (if the person's DNA isn't exactly complementary to the probe)
what is the unifying element between genomics, proteomics, metabolomics, and glycomics?
the ability to analyze massive amounts of data with a single experiment

global scale characterization of expression of different genes between healthy and diseased cells
what are the two types of microarrays?
commercial - system builds sequences on solid support using lithography

custom - PCR products applied to a glass slide
how are microarrays used?
the "probes" on the chip are screened with fluorescent-labelled "target" from 2 sources and the colors are compared

red - expressed in sample 1
green - expressed in sample 2
yellow/orange - expressed in both samples
what are microarrays?
gene chips

small solid objects with very high density of gene sequences which act as probes for target DNA
what is the problem with gene chips (genomics)?
gene chips show the RNA produced in a cell, however not all RNA is necessarily translated, so there is no guarantee that any RNA will be translated to protein
what is the benefit of proteomics?
allows high through-put sequencing of differentially detected proteins in 2-D gels

(can sequence several thousand proteins at once)
what method sequences the "spots" from a 2-D gel of proteins?
mass spectrometry
how is gain-of-function used to determine gene functionation?
expression vectors or RNA injections (into a single cell) are used to overexpress the gene
how is loss-of-function used to determine gene functionation?
eliminate expression of gene expression either in knockout mice or by antisense RNA, RNAi/siRNA, or dominant negatives
what are morpholino oligos (oligomers)?
oligonucleotide designed in antisense, modified to be more stable to exonuclease and endonuclease activities so that they stay bound to target RNA much longer, inhibiting translation
what are the two types of antisense technology to modify gene expression?
morpholino oligos
expression vector
what is shRNA?
short haipin RNA

synthetic miRNA
what is miRNA?
micro RNA

short RNA molecules that bind to complementary sequences on target messenger RNA transcripts, that usually results in translational repression and gene silencing
what is common to all miRNAs?
stem-loop structure

(which is cleaved away by DICER complex)
what is the function of DROSHA?
recognizes hairpin loop structure of pre-miRNA and transports the pre-miRNA out of the nucleus
by what process is miRNA regulatory of gene translation?
Drosha recognizes hairpin-loop structure which makes a pre-miRNA and transports it out of the nucleus

Dicer takes the loop structure off of the pre-miRNA and generates small RNA duplex

RNA duplex is separated by helicase and one of the strands targets its complementary RNA

the binding of miRNA with its complementary RNA targets that target for degradation via the RISC complex
what is the RISC complex?
RNA induced silencing complex

a complex of proteins which incorporates either a miRNA or siRNA to recognize complementary strands of mRNA and cleaves the mRNA

important for gene expression regulation and for defense agains dsRNA viruses
by what process is siRNA defensive?
mature siRNAs are incorporated in the RISC complex which degrades viral RNA after it has been cleaved by dicer and unwound by helicase
why are RNAi therapeutics useful?
they target RNA which encodes a specific protein for degradation

utilize miRNA to knock down the expression of a particular protein
what is somatic genetic engineering?
altering/replacing a gene in any cell except a gamete (affects only that individual)
what is germline genetic engineering?
altering/replacing a gene in a gamete (affects future generations)
how are inherited disorders targeted for gene therapy?
replacement or prophylactic introduction of non-defective gene

utilizes cells of the affected organ
how are cancers targeted by gene therapy?
expression of immune system modulating molecules (IL-2, TNF)

utilizes immune system cells
why are adeno-associated viruses the most beneficial vectors?
don't integrate into host genome
how are genetic treatments delivered in an ex vivo mode?
remove cells from affected tissue
introduce vector to cells
reinsert cells into affected tissue
how are genetic treatments delivered in an in vivo mode?
introduce vector directly into affected tissue, where it is taken up and expressed
what are the advantages of stem cells?
have the ability to grow and multiply (self-replenishment)

have the ability to differentiate into other cell and tissue types
how are stem cells classified in regards to the number of cell/tissue types they can differentiate into?
unipotent - one other cell/tissue type

multi-potent/pluripotent - several other cell/tissue types

totipotent - any/all other cell/tissue types
what is the most totipotent cell?
fertilized embryo
what are embryonic stem cells?
stem cells derived by in vitro culture of cells from inner cell mass of blastocyst

can develop into any cell type
what are adult stem cells?
stem cells derived from different niches in an adult (neural, hematopoietic, mesenchymal)

can develop only into cell types from the niche which they are derived from
what are induced pluripotent stem cells?
somatic cells induced to stem cells by introduction of vectors expressing genes

can develop into many cell types (more than adult stem cells), but not as many types as embryonic stem cells