Recombinant DNA Technologies (SINGER) Flashcards
What are some of the impacts on recombinant DNA technology on Medicine with the human genome?
the human genome project was completed
What is the impact on recombinant DNA technology on medicine involving genes?
Discovery of new genes and proteins produced
Cloning/isolating genes
study of gene function and regulation
profiling gene abnormalities
alteration/re-engineering of genes to transfer back into animals
What is the impact on recombinant DNA technology on medicine involving screening or preventative care?
producing vaccines and protein hormones, diagnositic confirmation in symptomatic patients, carrier testing for genetic disorders, prediction and response to treatment, population screening to predict future genetic disease.
Cleavage ofDNA at specific sites by restrcion endonucleases facilitates what?
the isolation and manipulation of genes
What facilitates the isolation and manipulation of genes?
Cleavage of DNA at specific sites by restriction endonucleases
What uses cloning vectors or PCR allowing single molecules to be copied to generate billions of identical molecules
DNA cloning
What is DNA cloning?
uses cloning vectors or PCR allowing single molecules to be copied to generate billions of identical copies
What is used to identify specific DNA + RNA sequences on the basis of binding to complementary nucleotide sequence?
Nucleic Acid Hybridization
What is nucleic acid hyridization used for?
used to identify specific DNA + RNA sequences on the basis of binding to complementary nucleotide sequence.
What is Sequencing of DNA fragments used for?
used to identify genes and deduce the amino acid sequence they encode.
What is used to identify genes and deduce the amino acid sequence they encode?
sequencing of DNA fragments
What is used to manipulate DNA sequences and reintroduce into viable organisms?
genetic engineering
Cleavage of DNA with restriction endonucleases digest the (blank) of DNA.
phosphodiester backbone
Each restriction enzyme has a specific (blank) recognition site.
palindromic
There are three types of cuttings produced by an endonuclease, what are they?
5’ overhang, 3’ overhang, blunt cutting
What endonucleases create 5’ overhang?
Pstl
What endonuclease create a 3’ overhang?
HindIII, EcoRI
What endonuclease creates a blunt cut?
Hpal
The (blank) ends of endonuclease cut nucleotide sequences have complementary base pairing.
cohesive or sticky
How does gel electrophoresis separate DNA?
by size (molecular weight)
DNA has a net (blank) charge.
negative
What type of gel does electrophoresis use?
agarose and polyacrylamide
In gel electrophoresis, are the larger DNA molecules or Smaller DNA molecules at the top?
larger
DNA molecules that differ in size by (blanK) nucleotide can be separated from each other in gel electrophoresis
one
IS the cathode positive or negative and is it at the top or bottom of gel electrophoresis?
cathode (-) and at top
After DNA is ran through gel electrophoresis it needs to be made visible through the use of what three things?
ethidium bromide, SYBR green, or 32P
What isolates DNA fragments and then propagates identical copies?
DNA cloning
What are double stranded circular molecules, they occur naturally in bacterial cells, are self-replicating and have small size?
vectors
What are 4 types of cloning vectors?
plasmid, phage, cosmid, BAC, YAC
What type of vector creates a high number of copies, physically isolates DNA and has a 10kb DNA limit?
plasmid
What type of vector infects bacteria, isolates DNA via phage packaging and has a DNA limit of 20kb?
Phage
What type of vector is based on F plasmid and physically isolates DNA, and has a DNA limit of 300kb
BAC
Why type of vector creates a high number of copies, isolates DNA via phage packaging and has a DNA limit of 48kb?
Cosmid
What type of vector has a orgin + centromere + telomere and physically isolates DNA and has a limit of 1 Mb?
YAC
How can you recognize a plasmid vector?
origin of repliction (ability for autonomous replication), multiple cloning site containing restriction sites to facilitate cloning of insert DNA,
Antibiotic resistance gene-selectable marker, small size for efficient transformation, a screening market to select for recombinants
using (blank) enable a research to insert a foreign DNA fragment into a plasmid vector?
restriction enzymes and DNA ligase
The plasmid is cleaved with a (blank) know to recognize a single site.
restriction enzyme
After your plasmid is cleaved with a restriction enzyme, what is used to cleave a fragment of foreign DNA generating fragments that have the same cohesive ends as the cleaved plasmid?
same restriction enzyme
The fragments of foreign DNA and cleaved plasmid DNA are incubated with DNA (blank) under conditions that favor base pairing between cohesive ends.
ligase
After restriction enzymes are used on plasmid and foreign DNA and then DNA ligase fuses these together, what does it generate?
a recombinant plasmid vector carrying the gene of interest
What kind of linkage is it when the foreign DNA is inserted into the plasma?
a covalent linkage
How is a recombinant DNA plasmid introduced into E. coli?
transformation
What are three screening procedures to detect a recombinant plasmid?
antibiotic selection (kanamycin, ampicillian resistance), blue/white screening (disruption of LacZ), size determination
An entire collection of clones constitutes a (blank)
library
What are the 2 types of libraries?
genomic DNA library, cDNA library
the cDNA library requires the conversion of (blank to blank). This conversion is call the (blank) step.
RNA to DNA
cDNA synthesis ste
what makes a DNA copy from an RNA strand?
reverse transcriptase
what kind of primer does a reverse transcriptase use?
oligo dT primer
What is the first step in making a cDNA library?
lyse cells and purify mRNA
What enzyme do you use to degrade RNA?
RNase H
What are the steps of creating cDNA?
start with lysed cells and purified mRNA, hybridize mRNA with poly-t primer which makes complementary DNA via reverse transcriptase, then degrade the RNA with RNase H and then synthesize a second DNA strand using DNA polymerase and the leftover RNA fragment as a primer
What is the main difference between genomic DNA clones and cDNA clones?
genomic DNA clones have introns, exons and transcriptional factors. cDNA has only exons of DNA or only coding regions
What can detect one molecule per cell and takes advantage of the fact that nucleic acid molecules like to create double strands.
Nucleic acid hybridization
(blank) reactions are very selective and specific. And can even detect differences in a single base pair.
hybridization
The specificity of nucleic acid hybridization can be altered by changing the stringency of the reaction, what 2 things can do this and why would you want to do this?
temperature and salt concentration.
You would want to do this because if you want a probe to bind to something that isnt compleletly complementary, you can alter stringency to force it to bind anyway.
What are the four types of hybridization processes?
in-situ
Southern Blot
Northern Blot
Microarray
A southern blot looks at what??
A northern blot looks at what?
a western blot looks at what?
DNA, RNA, protein
In situ hybridization can be within an (blank) cell, tissue or chromosme
intact
In situ hybridization reveals the distrubution of a particular (blank) molecule within a cell
DNA or RNA
In Situ Hybridization reveals the (blank) of a gene on a chromosome
position or location
To blot you need to denature your nucleic acid molecule and then run it on a gel then you transfer it onto membrane how?
with an electrical field or by gravity(most common)
in blottin,once you have your blot you will add your probe which will (blank) to your wanted sequence and then you will wash away unbound probe and expose to x-ray film
hybridize
What are the ways to examine cell protein interactions>
GST fusion protein pull-down assay Yease 2 hybrid fluorescence resonance energy transfer Regulated gene expression mutagenesis antisense technology RNA interference (RNAi pathway) siRNA
regulated gene expression utilizes what ?
repressor proteins and their substrates
Mutagenesis creates mutants that either lack a gene or have an altered version to look a gene expression and cellular function. What are three ways to do this?
1) primer with single base pair mutation is used to modify protein coding sequence
2) annealing to allow single base pair mismatch
3) mutation gets incorporated and results in protein with alterfucion
What does antisense technology do?
short DNA/RNA olionucleotides base pair to specific gene sequences
What effects does antisense technology have on target?
inhibition by blocking RNA splicing, prevents transport of mRNA antisense complex into the cytoplasm, Increase RNA degredation, Blocks initiation of translation
Thought to be exogenous in origin and Excised from long, complementary dsRNA and use argonaute protiens to support silencing functions
siRNA
Endogenously expressed Processed from a
stem-loop precursor use argonaute proteins
miRNA
What are two methods of producing transgenic mice?
1) targeting a gene in ES cells + Homoloous recombinations w/ locus deletion screening
2) pronucleus micro-injections
How does regenerative medicine works?
i. Inner cell mass is removed and growth continues in dish
ii. Culture conditions are changed in order to stimulate cells to differentiate into a variety of cell types such as skin cells, skeletal muscle cells and neural cells.
When is direct testing done?
when the gene of interest’s location on a chromosome is known. For example Cystic Fibrosis carrier testing
“ As soon as a chromosomal location for a disease phenotype has been established, (blank) analysis helps determine whether the disease phenotype is only caused by mutation in a single gene or mutations in other genes can give rise to an identical or similar phenotype”
genetic linkage
