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Uni Biology > Biotechnological Processes > Flashcards

Biotechnological Processes Flashcards

1
Q

Artificial DNA synthesis absolute requirements

A
  1. A pre-existing single stranded DNA template
  2. A pre-existing free 3’ hydroxyl group (on a primer)
  3. A protein catalyst (an enzyme)
    DNA polymerase
  4. dNTP precursors (building blocks)
    dNTP = dATP, dCTP, dGTP, dTTP
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2
Q

when can this reaction occur

A

inna test tube if the temperature, pH, and salt conditions meet the needs of the enzyme

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3
Q

what does PCR do

A

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

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4
Q

steps of PCR

A

Each PCR cycle involves three steps:
1.
Denaturation (high temperature separates DNA
strands)
2.
Annealing of primers (low temperature)
3. DNA synthesis (intermediate temperature)

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5
Q

what is Taq polymerase

A

Taq polymerase is thermostable DNA polymerase

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6
Q

what does each cycel do

A

double the amount of DNA

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7
Q

reverse transcription PCR (4)

A

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

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8
Q

Quantitative RT-PCR

A

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

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9
Q

Selectivity of Primers

A

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

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10
Q

what can be identified by using PCR (4) (4)

A

anything with nucleic acids

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11
Q

PCR applications (4) (4)

A

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

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12
Q

DNA fingerprinting (4)

A

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

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13
Q

(4)

A

Implicating a crime suspect, acquitting the wrongly
convicted
Paternity
Identifying the deceased
Population studies
Conservation biology
Prenatal testing

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14
Q

power of distrimination (4)

A

Ability to discriminate between different
individuals
* The larger the number of loci used, the
more powerful the ability to discriminate

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15
Q

1977: First generation: Sanger

A

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

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16
Q

ways to validate the quality of an assembly

A

N50
BUSCO
%Ns
% chimeric contigs
true pseudomolecules

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17
Q

N50

A

get all fragments, order them smallest to largest, get the one that is half the assembly size at N50

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18
Q

BUSCO

A

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

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19
Q

Sequencing a genome

A

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

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20
Q

Why a pangenome?

A

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
54

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21
Q

what is pangenome

A

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

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22
Q

define mRNA

A
  • intermediate form of
    information from nucleus to cytoplasm for processing
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23
Q

define rRNA

A

class of RNA found in
ribosomes, is essential for their function in protein
production

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24
Q

define tRNA

A

intermediary adapter molecule
between mRNA and amino acids during protein synthesis

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25
Q

define codon (4)

A

block of three DNA nucleotides corresponding to an amino acid

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26
Q

how is genetic code read (4)

A

genetic code is read in increments of three, read continuously

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27
Q

what does the addition or deletion of 1 or 2 nts do

A

shifts the genetic message

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28
Q

what does the addition or deletion of 3 nts do

A

will result in a protein that is normal aside from the addition/deletion

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29
Q

what are the three termination codons

A

UAA, UGA, UAG

30
Q

what is the codon used to signify the start of translation

A

AUG, methionine

31
Q

explain the phrase “code is degenerate”

A

meaning that some amino acids are specified by more than one codon

32
Q

what is the strongest evidence that all living things share a common ancestry

A

the fact that genetic code is practically universal

33
Q

difference with DNA replication and transcription

A

transcription does not require a primer (versus DNA replication which does)

34
Q

what are the two forms of RNA polymerase

A

core polymerase
holoenzyme

35
Q

what is holoenzyme

A
  • needed to accurately initiate synthesis
  • formed by the addition of (sigma symbol) (sigma) subunit
36
Q

things required for transcription

A
  • promoter
  • start site
  • terminator
37
Q

promoter

A

forms recognition and binding site for RNA
polymerase

38
Q

start site

A
  • actual site where RNA synthesis begins
39
Q

terminator

A

signal to end transcription

40
Q

WHAT IS THE RANSCRIPTIUON UNITTTITITIT

A

Region from promotor to the terminator is called the
transcription unit

41
Q

promoter

A
  • Found upstream of the start site

Not transcribed

42
Q

how does rna chain grow

A

RNA chain grows in the 5’-to-3’ direction as
ribonucleotides are added

43
Q

what is transcription bubble

A

Transcription bubble - contains RNA polymerase, DNA
template, and growing RNA transcript

44
Q

what occurs After the transcription bubble passes

A

After the transcription bubble passes, the now-transcribed
DNA is rewound as it leaves the bubble

45
Q

what is the RNA-DNA hybrid

A

RNA-DNA hybrid within the transcription bubble
dissociates
RNA polymerase releases the DNA
DNA rewinds

46
Q

Termination occurs at specific sites

A

Sequences the “stop” signal to RNA polymerase

47
Q

why is prokaryotic transcription unique

A

the coupling of transcription and translation

48
Q

explain the the coupling of transcription and translation

A

Prokaryotic transcription
is coupled to translation
mRNA begins to be
translated before
transcription is finished

49
Q

operon define it

A

Operon: A single mRNA may contain multiple genes

Grouping of functionally related genes

Encodes multiple enzymes for a pathway

Can be regulated together

50
Q
A

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

51
Q
A

RNA polymerase I transcribes rRNA

52
Q
A

RNA polymerase II transcribes mRNA and some snRNA

52
Q

mRNA modification

A

addition of a 5’ cap (methyl GTP is added to 5’ end)

addition of a 3’ poly-A tail

removal of introns

52
Q
A

RNA polymerase Ill transcribes tRNA and some other
small RNAs

52
Q
A

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

52
Q

do prokaryotes have introns

A
52
Q

exons

A

(expressed sequences) sequences that will be translated

53
Q

introns

A

(intervening sequences) non coding sequences (these will not be represented in the mRNA)

54
Q

single nucleotide polymorphisms

A
54
Q

how much of human genome has exons

A

1.0%-1.5% of the human genome is devoted to exons

55
Q

alternative splicing

A

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

56
Q
A

Transcriptome: all the RNAs produced from a genome

57
Q
A

Proteome: all the proteins produced from a genome

58
Q

ribosomes

A

Ribosomes are the key macromolecular machine involved in
translation, requires interaction with mRNA and tRNA to
synthesize proteins

59
Q

tRNA molecules

A

tRNA molecules can interact with mRNA and amino acids,
carry amino acids to the ribosome for incorporation into a
polypeptide

60
Q

Aminoacyl-tRNA synthetases

A

Aminoacyl-tRNA synthetases add amino acids to the
acceptor stem of tRNA

61
Q

Anticodon loop

A

Anticodon loop contains three nucleotides
complementary to mRNA codons (anticodon)

62
Q

wobble pairing of bases

A

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

63
Q
A

in eukaryotes the two processes are separated spatially and temporaly

64
Q
A

Mutations are defined as heritable change in the genetic
material, multiple types:

65
Q
A

A point mutation leads to single-nucleotide variation (SNV)
in populations (also called single-nucleotide polymorphisms
SNPs):

66
Q
A

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

Uni Biology (29 decks)

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