chapter 19 lecture 13/14 Flashcards
probe
DNA or RNA with a base sequence complementary to a sequence in the gene of interest, and with a radioactive or chemiluminescent molecule attached allows visualization
Southern blot
DNA
northern blot
RNA
western blot
protein
Polymerase chain reaction
amplifying DNA fragments
gene cloning
amplifying a specific piece of DNA via a bacteria cell
cloning vector
a replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell
PRC reaction
Taq polymerase: stable DNA polymerase at high temperature
- reverse transcription PCR
Reverse transcriptase PCR
important to get a copy of the coding sequence of the DNA
qPCR
incorporate a florescent probe that allows you to quantify how much code is present
Real-time PCR
quantitatively determining the amount of DNA amplified as the reaction proceeds
Multiplexing Real-time PCR
probes with different spectral wavelengths can be used to allow the quantitation of multiple targets in a single reaction
Gel electrophoresis
- dNA migrate to + electrode
- separation based on fragment length
- DNA cut with restriction enzyme
- gel soaked in ethidium bromide which sticks to DS DNA and fluoresces when exposed to UV light
restriction enzymes
recognizing and cutting DNA at specific nucleotide sequence
what do prokaryotes use to ward off viral parasites?
restrictions enzymes
Where do restriction enzymes come from?
exist naturally in bacteria, which use to prevent the entry of viral DNA
DNA fragments that are 500bp , 1000bp amd 2000 bp in length are separated by a gel electrophoresis. Which fragment will migrate farthest in the gel? a, 2000 bp fragments b. 1000 bp fragment c. 500 bp fragment d. all wllmigrate equal distance
c. 500 bp fragment
how do Northern and Western blotting differ fromsouthern blotting?
southern - used to transfer DNA from a gel to a solid medium
- Northern used to transfer RNA from a gel to a solid medium
- Western used to transfer protein from a gel to a solid medium
probe in southern and northern is a complementary sequence western is an antibody
cloning vector has what 3 characteristics
- an origin of replication : ensures vector replicated within the cell
- selectable markers: enable cells containing vector to be ID’d
- 1 or more unique restriction site into which a DNA fragment can be inserted
how is a gene inserted into a plasmid cloning vector?
gene and plasmid cut with same restriction enzyme and mixed together. DNA ligase is used to seal the nicks in the sugar backbone
Why is the use of a heat- stable DNA polymerase important to the success of PCR?
heat-stable DNA polymerase enzyme is important to the success of PCR because the first step of the reaction requires that the solution be heated to between 90 and 100 degrees C to separate the two DNA strands. Most enzymes are denatured at this temperature. with the use of a heat-stable polymerase, the enzyme can be added at the beginning of the reaction and will function throughout multiple cycles
briefly explain how synthetic probes are created to screen a DNA library when the protein encoded by the gene is known
with the use of the genetic code and the amino acid sequence of the protein, possible nucleotide sequences that might encode the protein, taking into consideration synonymous codons is used to probe the library. to minimize the number of sequences required, a region of the protein that has a relatively little degeneracy in its codons is selected
how are candidate genes identified by positional cloning evaluated to determine whether they encode the phenotype of interest
the expression pattern of the gene can be examined and the coding region of copies of the gene from individuals with the mutant phenotype can be compared with the coding region of wild-type individuals
in the dideoxy sequencing reaction, what terminated DNA synthesis at a particular base
a. the absence of a base on the ddNTP halts tje DNA polymerase
b. the ddNTP causes a break in the sugar-phosphate backbone
c. DNA polymerase will not incorporate a ddNTP into the growing DNA strand
d. the absence of a 3’-OH group on the ddNTP prevents the addition of another nucleotide
d. the absence of a 3’-OH group on the ddNTP prevents the addition of another nucleotide
how are microsatellites detected?
by using PCR with primers that flank the region containing tandem repeats
DNA library
a collection of clones containing all the DNA fragments from one source
creating a genomic library
collection of the total genomic DNA from a single organism . DNA is stored in a population of identical vectors, each containing a different insert of DNA
cDNA libraries
consisting only of those DNA sequences that are transcribed into mRNA
In Situ Hybridization
DNA probes used to determine the chromosomal location and to visualize a gene while it is in a cell
FISH, Florescense in Situ Hybridization
Positional cloning
isolating genes on the basis of their position on a genetic map
a geneticist is interested in immune function induces random mutations in a number of specific genes in mice and then determines which of the resulting mutant mice have impaired immune function. This procedure is an example of:
a. forward genetics
b. reverse genetics
c. both forward and reverse genetics
d. neither forward nor reverse genetics
b. reverse genetics
forward begins with phenotype and detects and analyzes the genotype that causes the phenotype.
reverse begins with gene sequence and through analysis determines phenotype
what is the advantage of using the neo gene to disrupt the function of a gene in knockout mic?
a. the neo gene produces an antibiotic that kills unwanted cells
b. the neo gene is the right size for disabling other genes
c. the neo gene provides a selectable marker for finding cells that contain the disabled gene
d. the neo gene produces a toxin that inhibits transcription of the target gene
c. the neo gene provides a selectable marker for finding cells that contain the disabled gene
chromosome walking
technique used after positional cloning to more precisely locate the locus by using additional molecular markers
- progress from neighboring genes to linked clones, one of which might be gene of interest
chromosome jumping
a related technque to walking that allows one to move from more distantly linked markers to clones that contain a sequence of interest
cystic Fibrosis
gene located by positional cloning
how?
1. clones from region isolated by walking and jumping
2. analysis if DNA sequences within clones revealed 4 candidate genes
3. additional studies eliminated 3 of the candidate genes
4. DNA sequencing revealed the presence of a 3 bp deletion in the gene of the CF patient
What test provided evidence that the candidate gene isolated was in fact the gene that caused Cystic fibrosis?
Northern Blot
Sanger Sequencing
dideoxy-sequencing
set up rxn that will replicate a single strand of DNA provided you have all the components
- If include small % of dideoxy NT it will terminate when if gets one of them
○ Incorporate at random when gets a G
§ So get a series of fragments that have a G at the position
Each labeled with a p32 on those C’s
sanger sequecning with florescent labels
each NT has a color coded to it 4- single strand DNA added and rxn is carried out
- products denatured, loaded onto well, electrophoresis, fragments migrate, gel read by laser and peaks of color go to computer, into read directly into computer and have your sequence
pyrosequencing
sequencing of ssDNA by synthesizing the complementary strand one base pair at a time and detecting which base pair is actually added at each step
- produces a light when the base pair is added and the light is recorded
illumina sequencing
bind fragments, ligate on complementary and then primer that will allow expansion of fragment and as extends have tagged NT that when admitted can see
- allows cost effective high resolution sequencing
nanopore sequencing
sequence determination of single molecules of nucleic acids that electrophoretically pass through nanoscale pores
- 3rd generation sequencing
Metagenomics
involves IDing organisms present in the intestine, sewage, a body of water, dirt, debris filtered from a ir, or anywher with living organisms
- alls determination of types of microbes may be present in a microbiome
microsatellites
STRs
-variable number of copies of repeat sequences possessed by many organisms
- detected by PCR
- fragments represented as peaks in a graph
0 each person has a unique compliment of microsatellites = DNA fingerprint
application of DNA fingerprinting
also carried out on mitochondrial DNA
- requires a source of DNA with known identity
ex toothbrush or hairbrush
humanized mouse
a mouse carrying functional human genes, cells, tissues, and/or organs
- usually accept dues to lack of host immunity
Transgenic animals
an organism permanently altered by the addition of a DNA sequence to its genome
What are some concerns about genetically engineered crops?
- ecological damage caused by introducing novel organisms into the environment
- negative effects of transgenic organisms on biodiversity
- possible spread of transgenes to native organisms by hybridization 4 health effects of eating genetically modified foods
what is the difference between somatic gene therapy and germ-line gene therapy?
somatic only modifies genes in somatic tissue and can’t be inherited - germ-line alters genes in germ-line cells and will be inherited
oligonucleotide-directed mutagenesis
used to study gene function when appropriate restriction sites are not available
site0directed mutagenesis
mutations induced at specific location
creating random mutations
radiation , chemical mutagens, and transposable elements are used to create mutations for genetic analysis
knock out mice
mice in which a normal gene has been not just mutated by fully diabled
- useful in determining the function of a gene
silencing with RNAi
design an siRNA that will be recognized and cleaved by DICER (protein that processes siRNAs)
- sequence must be unique to target mRNA so that the siRNA will not inhibit nontarget mRNAs
- then siRNA synthesized
- cloned in plasmid between 2 strong promoters a
- produced into dsRNa recognized by DICER
and be able to deliver to cells
DICER
protein that processes siRNAs
application: using RNAi for the treatment of human disease
- high cholesterol
- shown to reduce the levels of ApoB and blood cholesterol in nonhuman primates
Gene therapy
set of strategies that modify the expression of an individual’s genes or that correct abnormal genes.
CRISPR/Cas system
prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages
- cut exogenous elements by delivering the Cas9 protein and appropriate guide RNAs into a cell, the target organism’s genome can be cut at any desired location
- recognizes nonself
- can incorporate a linker fragment to help healing but sequence is cut up and own DNA healing mechanisms heals the space
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