Molecular Biology techniques for cell biology Flashcards
module 2
What is polymerase chain reaction (PCR) widely used for?
to clone genes
how does polymerase chain reaction work?
- must know the sequence of DNA what to amplify
- Gene specific primers complementary to gene of intreats amplify sequence
- amplified DNA can be cloned
what are restriction fragments used for?
used to experimentally for recombinant DNA technology
what are the steps for restriction fragments?
- DNA from 2 sources treated with restriction enzyme to generate fragments with sticky ends
- fragments mixed together under conditions that favour base pairing btwn sticky ends
- cut molecules are sealed together by DNA ligase
what is the basis of restriction enzyme job?
to cut and recombine DNA
what does restriction endonuclease do?
cut DNA molecules at specific internal sites
what is the recognition sequence called in restriction endonucleases?
restriction site
what is the formation of the restriction site?
palindrome (antiparallel)
when restriction enzymes cut at the same point, what does it make?
blunt ends
how do restriction enzymes make sticky cuts?
cut at staggered sites
what does recombinant DNA technology allow?
allows scientists to create recombined DNA molecules with DNA from different sources
how are proteins studied through recombinant DNA technology?
can be studied and manipulated indirectly by manipulating DNA sequences that encode them
what is DNA cloning?
generation of many copies of a specific DNA sequence
what is DNA transformation?
process of introducing DNA into the cell
what does gel electrophoresis allows?
allows DNA to be separated by size
how does gel electrophoresis work?
DNA molecules are negatively charged therefore will migrate towards the anode (positively charged electrode)
what is the DNA detected with in gel electrophoresis?
ethidium bromide (stain), can see under UV light
what does DNA sequencing make possible?
to analyze the entire genome of an organism
what does genome wide sequence analysis do?
gives variety of types of information
what is pyro sequencing?
sequencing through light
how does pyro sequencing work?
four labeled nucleotides and firefly enzyme luciferase
incorporation of correct nucleotide on strand releases pyro phosphate which releases light when interacts with luciderase
what is illumination sequencers?
sequencing through fluorescents
what does specializes dNTPs do?
tag a unique fluorescent signal for each base
what is a reverse terminator do?
blocks the addition of new bases
what happens when a base is added with Illumina sequencers?
it can be identified in real time by florescence emissions
when a base is identified in illumina sequencers, what is the next step?
the terminator is removed, another base added and identified
what is third generation sequencing?
nano pre sequencing
what is nano pore sequencing?
produces long reads but doesn’t have a DNA synthesis step
what are the steps to nano pore sequencing?
- protein pores embedded in artificial membrane
- single stranded DNA goes through, disrupts electrical field
- each base disrupts the electrical field differently (allows base identification)
how are bases identified through nano pore sequencing?
when base disrupts electrical field, it has own electrical field signature
what does bioinformatics do?
helps decipher genomes
how does Bioinformatics work?
merges computer science with bio to organize sequencing and other data
what is an exome?
part of genome that contains exons (encoded proteins)
what is transcriptone?
the entire set if RNA molecules produced by a genome
what is a proteome?
all the proteins produced by an organism. it’a more complex than the genome
what is the percent of the genome all humans share?
99.7%
what is the percent of the genome that varies person to person?
0.03%
what does inherited diseases result from?
mutations in single loci (genes)
what are multi genetic diseases?
many different genes affect the likelihood that the disease will arise
how is RNA analyzed?
RT-PCR
RNA-SEQ
what does RT-PCR help you know?
necessary to know when and where RNAs are expressed
how does RT-PCR work?
cDNA is made from RNA through reverse transcription
cDNA amplified by PCR using primers
what is RT-PCR for?
indirectly determine whether an mRNA present in a sample
what is another name for RNA-SEQ (RNA sequencing)
also called whole transcriptome shotgun sequencing
where is the data assembled from RNA-SEQ?
bioinformatics tool
what is transcrtptomics?
study of the quantities and functions of all the RNAs present in a particular gene
how are proteins analyzed?
SDS polyacrylamide gel electrophoresis
western blotting with antibodies
dissecting protein function via mutations
what is SDS polyacrylamide?
used to separate proteins with heat and anionic detergent SDS
what are the steps of SDS polyacrylimide gel electrophoresis?
- proteins are solubilized
- loaded into wells of gel
- electrical potential is applied (anode at bottom)
- rate of movement related to their size (small travel fast)
- the gel is stained with dye to visualize proteins
how are specific proteins detected?
through western blotting with antibodies
what are the steps of western blotting with antibodies?
- protein mixture is solubilized and separated by SDS PAGE
- then blotted into membrane using electrical current
- blot incubated with primary anti-bodies and then secondary antibodies
- secondary attached to enzyme can be detected by coloured precipitate or through chemiluminescence
what is dissecting protein function via mutations?
pieces of DNA encoding a protein can be deleted, leaving DNA that encodes only part of protein
what does examining the protein fragment do in dissecting protein function?
helps determine which parts of the protein are important for the function
what is site-directed mutagenesis?
single base can be mutated so the resulting protein is altered
how is gene function analyzed?
genetic engineering
crispr genome editing
what is genetic engineering?
genetic engineering of proteins to in living organisms requires engineering the organism
what is a transgenetic organism contain?
foreign pieces of DNA that can be passed to subsequent generations
what is the process to create organisms called transgenesis?
- direct injection of bombardment
- infection (in plant bio technology)
what is crispr genome editing?
technologies to alter the genomes of cells and organisms collectively involve genome editing
how does CRISPR work?
sequences are used to target complexes to certain genomic sites were the genomic DNA is directly altered
what happens to double stranded breaks in CRISPR genome editing?
they are induced then repaired
what is the result of CRIPR genome editing?
the result id removal of the DNA or replacement of normal DNA with another sequence
what is the molecular biology applications of CRISPR genome editing?
guid RNA acts along with crRNA and CRISPR associated protein (Cas 9) to introduce double stranded breaks on foreign DNA
