Biotechnology Part 1 Flashcards
what does DNA amplification do? Why?
it create millions of copies of a specific DNA sequence to increase the amount of DNA
what are the materials in PCR?
- DNA template
- nucleotides
- DNAP (Taq polymerase)
- DNA primers
what are the 3 steps of the PCR cycle?
- Denaturation
- Annealing
- Extension
what is denaturation in the PCR cycle
temperature is increased to separate DNA strands
what is the annealing in the PCR cycle
temperature is decreased to allow primers to base pair to complementary DNA template
what is the extension in the PCR cycle
plymerase extends primer to form nascent DNA strand
describe a primer in the PCR cycle?
- a synthetic sequence (artificial)
- single stranded DNA
- provides the 3’ OH for polymerization
- 2 primers used for complementary to each end of the target region of the DNA
- defines length of target DNA to be replicated
Why is Taq used in the PCR cycle?
the temp of the PCR denatures the enzymes meaning they don’t work so something else must be used
what was done before Taq was used?
- 3 water baths at 3 temperatures with a re-addition of DNAP at the beginning of each synthesis step
- tedious and time consumming as it had to be done manually all day
describe the thermal cycler
has a plate that heats and cools in a process that takes 1-2 hours
what is used for unwinding compare natural DNA replication and PCR replication.
natural: Ori on DNA template + helicase
PCR: template + heat
what is used for priming? compare natural DNA replication and PCR replication.
natural: RNA primer + primase
PCR: DNA primers x2 + annealing
what is used for elongation? compare natural DNA replication and PCR replication.
natural: nucleotides + DNAP
PCR: nucleotides + Taq polymerase
what is used for termination? compare natural DNA replication and PCR replication.
natural: end of chromosome OR meets another replication bubble
PCR: end of DNA template or a change in temp
at which cycle do you get the correctly sized target sequence?
2 round of replication
what does endonuclease do?
breaks phosphodiester bonds within a nucleotide chain
what is a restriction enzyme?
a biological ‘scissor’
what is the function of a restriction enzyme
acts like the immune system of bacteria to protect it against DNA from other organisms
what does a restriction enzyme do in bacteria?
recognizes short nucleotide sequences in the foreign DNA - cuts cvalent phosphodiester bonds of both strands of DNA rendering foreign DNA harmless
restriction site
sequene recognized and cut by restriction enzyme
what does palindromic mean in bio?
same sequence on complementary strand in opposite orientation
what is a restriction fragment?
pieces of DNA created by restriction enzymes
what is a sticky end
a signle-stranded end of the restriction fragment
what is a blunt end
straight ends without any single-stranded regions
how does naming restriction enzymes work?
1 - first letter of first word in organism name
2 - first two letters of second word in organism name
3 - stran
4 - roman numeral of enzyme in this strain
recombinant DNA
genes from two different sources (often different species) combined into one molecule - any DNA cut with the same restriction can be ligated together because they have the same sticky ends that are complementary
plasmid
extisting circular bacterial DNA
what are the general steps of forming a recombinatant
- restriction enzyme digestion of plasmid and gene of interest
- hybridization of matching sticky ends on gene of interest and plasmid
- DNA ligase seals gene of interest with plasmid
genome
an organisms’ complete set of DNA
human genome project
is an international collavorative research effort 1998-2003 to sequence and map all genes in human beings
why does the CRISPR (Cas 9 system exist)
naturally as the bacteria’s immune system to destroy phages by recognizing foreign DNA and making a double stranded cut at that DNA
how does the CRISPR mechanism work
- CRISPR locus is transcribed and translated to make the CRISPR/CAs9
- CRISPR RNA targets Cas9 to specific complementary region on foreign DNA
- Cas9 makes a double stranded break at that location
what is the CRISPR/Cas9 structure?
ribonucleoprotein complex with CRISPR RNA and Cas9 endonuclease
what are the two main parts of the CRISPR locus
repeats and spacers
what does CRISPR stand for?
clustered regularly interspaced short palindromic repeats
what are the characteristic of a CRISPR locus
- regularly spaced
- short sequence
- identical
- palindromic
what are the two main steps in CRISPR/Cas9 mechanism
A. Immunization
B. Adaptive/Acquired Immune Response
what are the steps in the CRISPR/Cas9 mechanism
A. Immunization
1. acquire foreign DNA
2. Transcribe into RNA
3. process (Cut) RNA
4. incorporate into Cas9 protein
B. adaptive/acquired immune response
1. Targeting by cRNA
2. Interferene/Inactivation
What is the 1st step of the CRISPR/Cas9 mechanism
acquiring foreign DNA
describe acquiring foreign DNA
- bacteria would get infected by viruses
- sometimes bacteria survive infection and takes the viral DNA placing it within the CRISPR locus (spacer)
- is followed by a new repeated sequence
what is a repeat?
palindromic sequence same sequence repeated between each spacer
What is the 2nd step of the CRISPR/Cas9 mechanism
transcribe to RNA
What is transcription to RNA in CRISPR
the CRISPR locus transcribed into RNA called pre-cRNA, which causes palindromic nature of the repeat forms hairpin loops
what is the third step of the CRISPR/Cas9 mechanism?
Processing (cutting) RNA
Describe the processing of RNA
pre-cRNA cut into pieces forming crRNA with each one containing one spacer
what is the fourth step of the CRISPR/Cas9 mechanism? Describe it
incorporating into Cas9 protein - the crRNA is placed into Cas9 protein
How does Adaptive/Acwuired Immunity work?
if the virus tries to infect the cell again, the crRNA/Cas9 already exists SOOOO:
- the crRNA binds to the complmentary viral DNA
- causes a confirmational change in Cas9 activating it’s endonuclease acitivity
- Cas9 makes a double stranded blunt end that breaks and destroys the viral DNA
What is the ‘power’ of CRISPR /Cas9
you can manipulate the crRNA sequence to target whatever you want to be cut by Cas9
what is gRNA? what does it do?
guide RNA - replaces the crRNA with an RNA sequence of choice that is complementary to a desired DNA region or gene of interest - it will direct the Cas9 complex to the target site
what is PAM? what does it do?
protospare Adjacent Motif a sequence where N is any base
- Cas9 recognizes and binds to any PAm and unwindes that region of DNA
how does the Cas9 Endonuclease work?
if the exposed signle stranded DNA region unqound by Cas9 is complementary to the gRNA they will base pair with causes a conformational change in Cas9 activating its endonuclease activity, which results in a double stranded break at a site upstream of PAM
how does CRISPR/gRNA apply to gene editing?
gRNA sequence can be used, then any DNA region can be targeted and cut by Cas9, making gene editing more precise and less time-consuming
what are the two main repair mechanisms?
HDR: homology directed repair
NHEJ: non-homologous end joining
what is gene editing - repair by insertion
placement of a new DNA sequence at the targeted region
describe homology directed repair
cell with cut DNA is subjected to a large dose of the donor template DNA and natural DNA-repair mechanisms of the cell will insert the desired genetic material
what does the donor DNA sequence consist of?
- desired sequence
- DNA homologous to the blunt ends of the cut DNA flanking the desired sequence
what is the power of Power of CRISPR & HDR
you can insert any DNA sequence you want at any specific location
describe NHEJ
double-stranded breaks are ligated back together without the need for a homologous DNA template, which can result in missing bases
what is the Power of CRISPR and NHEJ
due to the error prone nature of NHEJ, you can target any gene and inactivate it
what is gel electrophoresis?
size separation of nucleic acids by moving them through a gel medium using electric current
how is the gel in gel electrophoresis made?
- liquid solution of gel medium is poured into a mold and allowed to set and solidify
- a cone is placed at one end of the gel, when removed it creates the wells where DNA is loaded
describe the liquid buffer in the gel
- contains ions to allow for flow of electricity
- prevents the gel from overheating and dryingout
what are the two gel mediums?
agarose and polyacrylamide
describe agarose
- seaweed extract
- lower resolution resolving power and
- separation of nucleic acid
describe polyacrylamide
- artificial polymer
- higher resolution resolving power
- nucleic acid and protein separation abilities
resolving power
the more precisely closely sized DNA fragments can be distinguished
describe the movement of DNA within the gel
DNA is negatively charged due to the phosphates so it will move toward the positive electrode
SHORT:
- moves through gel easily and travels further
LONG:
- moves through gel slower and not as far as the size makes it get caught int he web of polymers
what are the components of the loading dye
glycerol and coloured dyes
what is the purpose of glycerol in the loading dye?
- adds density to DNa sample
- so makes it sink down to the bottom of the well when placed int he gel
- DNA isn’t as easily lost in the buffer or spilled
what is the purpose of coloured dyes?
- used for tracking distance travelled on the gel by DNA since DNa is invisble
- indicates when it is time to turn off the gel electrophoresis
what is ethidium bromide? Describe it.
- a chemical that binds to the DNA
- makes DNA visible
- not good for the body - carcinogenic
- glows under UV light
Describe SYBR Safe
not carcinogenic and glows green
