Biotechnological Processes Flashcards
Artificial DNA synthesis absolute requirements
- A pre-existing single stranded DNA template
- A pre-existing free 3’ hydroxyl group (on a primer)
- A protein catalyst (an enzyme)
DNA polymerase - dNTP precursors (building blocks)
dNTP = dATP, dCTP, dGTP, dTTP
when can this reaction occur
inna test tube if the temperature, pH, and salt conditions meet the needs of the enzyme
what does PCR do
Mimics DNA replication to produce millions of copies of a
DNA sequence
Allows the amplification of a small DNA fragment using
primers that flank the region
steps of PCR
Each PCR cycle involves three steps:
1.
Denaturation (high temperature separates DNA
strands)
2.
Annealing of primers (low temperature)
3. DNA synthesis (intermediate temperature)
what is Taq polymerase
Taq polymerase is thermostable DNA polymerase
what does each cycel do
double the amount of DNA
reverse transcription PCR (4)
PCR is performed on cDNA made from mRNA
Called reverse transcription PCR (RT-PCR), it:
Allows creation of recombinant DNA containing only the
exons of genes
Allows study of the structure and function of gene products
Can be used to determine relative levels of gene
expression in cells/tissues
Quantitative RT-PCR
Reverse transcription quantitative PR (RT-qPCR)
involves isolating mRNA, converting to cDNA using RT,
then using PCR to amplify specific cDNAs
Amount of DNA produced can be measured in real time by
the PCR machine
Can be quantitated using DNA-binding dyes or DNA-
binding probes
Selectivity of Primers
Primers bind to their complementary sequence on the
target DNA
A primer composed of only 3 letter, ACC, for example, would be
very likely to encounter its complement in a genome. Four to the
power of 3 = 64
As the size of the primer is increased, the likelihood of, for
example, a primer sequence of 25 base letters repeatedly
encountering a perfect complementary section on the target DNA
become remote. Four to the power of 25
1,125,899,906,842,624
what can be identified by using PCR (4) (4)
anything with nucleic acids
PCR applications (4) (4)
Infectious agents (bacteria, viruses, fungi) contain nucleic acids, and can therefore be identified by PCR
Only primers for pathogen-specific genes required RNA viruses are more complicated as requires RT-PCR and handling of more delicate RNA molecules
Diagnostic kits have been developed to be faster and more accurate in response to COVID-19 pandemic
DNA fingerprinting (4)
Need to identify an individual based on a small amount of
tissue or bodily fluids
Takes advantage of short tandem repeats (STRs) that vary
among individuals
Population is polymorphic for these markers
PCR primers flank a region known to contain an STR
Using several probes, probability of identity can be
calculated or identity can be ruled out
(4)
Implicating a crime suspect, acquitting the wrongly
convicted
Paternity
Identifying the deceased
Population studies
Conservation biology
Prenatal testing
power of distrimination (4)
Ability to discriminate between different
individuals
* The larger the number of loci used, the
more powerful the ability to discriminate
1977: First generation: Sanger
Fragment DNA
Clone into plasmid and amplify
Sequence using dNTP + labelled
ddNTPs (stops reaction)
* Run capillary electrophoresis/gel and
“read” DNA code
*
Low output, long reads (~800-1200 nt),
high quality
ways to validate the quality of an assembly
N50
BUSCO
%Ns
% chimeric contigs
true pseudomolecules
N50
get all fragments, order them smallest to largest, get the one that is half the assembly size at N50
BUSCO
all these genes are present in all these organisms, so if they aren’t present in your assembly its not complete, which kinda works but genes maybe just be missing in that species/organism
Sequencing a genome
Generate data
* Reduce complexity?
BACs
Isolated chromosomes
Whole genome shotgun
* Data types
Sanger
Illumina
» Paired end
» Mate paired
» Long mate paired
Pacific Biosciences
- Oxford Nanopore
Why a pangenome?
A reference genome does not represent the diversity
of a species
* PAV genes are responsible for important traits
* Need to know gene content for genome editing
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what is pangenome
its not the genome of an individual, its the genome of a species, this idea was developed in bacteria because theres a lot of presence and absence variation in bacteria because they keep on exchanging DNA
define mRNA
- intermediate form of
information from nucleus to cytoplasm for processing
define rRNA
class of RNA found in
ribosomes, is essential for their function in protein
production
define tRNA
intermediary adapter molecule
between mRNA and amino acids during protein synthesis
define codon (4)
block of three DNA nucleotides corresponding to an amino acid
how is genetic code read (4)
genetic code is read in increments of three, read continuously
what does the addition or deletion of 1 or 2 nts do
shifts the genetic message
what does the addition or deletion of 3 nts do
will result in a protein that is normal aside from the addition/deletion
what are the three termination codons
UAA, UGA, UAG
what is the codon used to signify the start of translation
AUG, methionine
explain the phrase “code is degenerate”
meaning that some amino acids are specified by more than one codon
what is the strongest evidence that all living things share a common ancestry
the fact that genetic code is practically universal
difference with DNA replication and transcription
transcription does not require a primer (versus DNA replication which does)
what are the two forms of RNA polymerase
core polymerase
holoenzyme
what is holoenzyme
- needed to accurately initiate synthesis
- formed by the addition of (sigma symbol) (sigma) subunit
things required for transcription
- promoter
- start site
- terminator
promoter
forms recognition and binding site for RNA
polymerase
start site
- actual site where RNA synthesis begins
terminator
signal to end transcription
WHAT IS THE RANSCRIPTIUON UNITTTITITIT
Region from promotor to the terminator is called the
transcription unit
promoter
- Found upstream of the start site
Not transcribed
how does rna chain grow
RNA chain grows in the 5’-to-3’ direction as
ribonucleotides are added
what is transcription bubble
Transcription bubble - contains RNA polymerase, DNA
template, and growing RNA transcript
what occurs After the transcription bubble passes
After the transcription bubble passes, the now-transcribed
DNA is rewound as it leaves the bubble
what is the RNA-DNA hybrid
RNA-DNA hybrid within the transcription bubble
dissociates
RNA polymerase releases the DNA
DNA rewinds
Termination occurs at specific sites
Sequences the “stop” signal to RNA polymerase
why is prokaryotic transcription unique
the coupling of transcription and translation
explain the the coupling of transcription and translation
Prokaryotic transcription
is coupled to translation
mRNA begins to be
translated before
transcription is finished
operon define it
Operon: A single mRNA may contain multiple genes
Grouping of functionally related genes
Encodes multiple enzymes for a pathway
Can be regulated together
RNA polymerase I transcribes rRNA
RNA polymerase II transcribes mRNA and some snRNA
RNA polymerase Ill transcribes tRNA and some other
small RNAs
Each RNA polymerase recognizes its own promoter
RNA polymerase I transcribes rRNA
RNA polymerase II transcribes mRNA and some snRNA
mRNA modification
addition of a 5’ cap (methyl GTP is added to 5’ end)
addition of a 3’ poly-A tail
removal of introns
RNA polymerase Ill transcribes tRNA and some other
small RNAs
Core promoter and RNA Pol Il
Initiation of transcription at Pol I promoters
Requires a series of transcription factors
Elongation complex factors
Additional factors including chromatin-remodeling
complexes
do prokaryotes have introns
exons
(expressed sequences) sequences that will be translated
introns
(intervening sequences) non coding sequences (these will not be represented in the mRNA)
single nucleotide polymorphisms
how much of human genome has exons
1.0%-1.5% of the human genome is devoted to exons
alternative splicing
The ratio of genes to transcripts to proteins is not 1:1:1
Alternative splicing, process of a single primary transcript
being spliced into different mRNAs by including different s ets
of exons, can account for deviation from balanced ratio
Current estimates are ~20,000 human protein-encoding
genes and 80,000+ protein-encoding transcripts
Transcriptome: all the RNAs produced from a genome
Proteome: all the proteins produced from a genome
ribosomes
Ribosomes are the key macromolecular machine involved in
translation, requires interaction with mRNA and tRNA to
synthesize proteins
tRNA molecules
tRNA molecules can interact with mRNA and amino acids,
carry amino acids to the ribosome for incorporation into a
polypeptide
Aminoacyl-tRNA synthetases
Aminoacyl-tRNA synthetases add amino acids to the
acceptor stem of tRNA
Anticodon loop
Anticodon loop contains three nucleotides
complementary to mRNA codons (anticodon)
wobble pairing of bases
There are fewer tRNAs than codons
Wobble pairing allows less stringent pairing between the 3’
base of the codon and the 5’ base of the anticodon
This allows a lower number of tRNAs to accommodate all
codons
in eukaryotes the two processes are separated spatially and temporaly
Mutations are defined as heritable change in the genetic
material, multiple types:
A point mutation leads to single-nucleotide variation (SNV)
in populations (also called single-nucleotide polymorphisms
SNPs):
Base substitution - substitution of one base for another, two
categories:
Transition if purine-purine or pyrimidine-pyrimidine
mutation
Transversion if purine-pyrimidine or vice-versa mutation
