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
