Section 3 - Module 12/13 Flashcards by Kierra McCaffrey

Why clone DNA in living cells?

1) before PCR, cloning was the only way to copy (amplify) DNA sequences
2) to make more DNA with high fidelity for further study or manipulation
3) to produce substances of scientific or commercial value from genes
4) to modify the genomes of plants or animals to introduce new, desired traits

How well did you know this?

Not at all

Perfectly

Do living cells or PCR copy DNA with more accuracy?

Living cells

How well did you know this?

Not at all

Perfectly

What does cloning require?

a vector

How well did you know this?

Not at all

Perfectly

What is a commonly used cloning vector?

Plasmid

How well did you know this?

Not at all

Perfectly

Why are plasmids common vectors?

stable, self-replicating molecule, circular DNA molecule, there is a origin of replication, unique retrusion sites, has selectable markers

How well did you know this?

Not at all

Perfectly

What is pUC 19?

A commonly used bacterial vector

How well did you know this?

Not at all

Perfectly

What makes up pUC 19?

portion of the lacZ+ gene, with a restriction site ‘linker’ that contains numerous unique restriction enzyme cut sites, and antibiotic resistance gene

How well did you know this?

Not at all

Perfectly

what is the pUC 19 antibiotic resistance gene?

ampR (for amplicon)

How well did you know this?

Not at all

Perfectly

What is meant my ‘unique’ restriction enzyme cut site?

sites found no where else on the plasmid

How well did you know this?

Not at all

Perfectly

What is ‘competent’ bacteria?

E. coli made receptive to transformation by chemical or electrical treatment

How well did you know this?

Not at all

Perfectly

What is the lacZ gene in the transforming bacteria?

lac Z-, meaning they lack the portion of lac Z gene that is present in the plasmid

How well did you know this?

Not at all

Perfectly

What happens to bacteria with no plasmid (transforming bacteria & selecting for recombination)?

do not grow since they have no antibiotic resistance

How well did you know this?

Not at all

Perfectly

What do bacteria with non-recombinant plasmid produce?

beta-galactosidase, resulting in blue colonies

How well did you know this?

Not at all

Perfectly

What do bacteria with recombinant plasmid produce?

do not produce beta-galactosidase resulting in white colonies

How well did you know this?

Not at all

Perfectly

What do bacterial expression vectors include?

operon and regulatory sequences (allows expression of genes in bacteria)

How well did you know this?

Not at all

Perfectly

what is cDNA

combined growth hormone gene

How well did you know this?

Not at all

Perfectly

What is a measure taken to reduce the risk of transgenic salmon interbreeding with wild salmon?

Farmed salmon will be triploid (triploid females are sterile)

How well did you know this?

Not at all

Perfectly

What is Rhizobium radiobacter (formerly known as agrobacterium tumefaciens) do?

Naturally transforms the DNA of higher plants

How well did you know this?

Not at all

Perfectly

What does Bacillus thuringiensis produce?

a protein, Bt toxin, lethal to many insects

How well did you know this?

Not at all

Perfectly

Desirable features of Bt toxin?

Toxicity specific to some insects, not toxic to humans & other animals, biodegradable

How well did you know this?

Not at all

Perfectly

What is routinely used to check success of experiments?

PCR

How well did you know this?

Not at all

Perfectly

What is routinely used in combination with RE cleave or PCR to check the success of cloning/transformation experiments?

Gel electrophoresis

How well did you know this?

Not at all

Perfectly

When was “Celera Genomics will describe
the first assembly of the human genetic
code from the whole genome shotgun
method” said?

during the 1st draft of human genome accouchement under president Bill Clinton said by Venter

How well did you know this?

Not at all

Perfectly

What is issue with shotgun sequencing?

problems with repetitive DNA sequences

How well did you know this?

Not at all

Perfectly

How to fix issue of repetitive DNA sequences and shotgun sequencing method?

long-read sequences (like nanopore)

What are the advantages of long reads?

They help with the assembly and alignment of short reads

Base calling

greater read depth gives more confidence a base is accurately

Resequencing

When additional copies of a genome are sequenced

What is the transcriptome?

sequences sorted in to different genes

Applications of draft of human genome?

1) genome assembly by shotgun sequencing 2) transcriptomics, gene expression analysis 3) identifying species (DNA barcoding; the inventory of animal life) 4) studying microbiomes 5) environmental DNA (eDNA)

What DNA is most widely used for identifying animal species?

mitochondrial DNA (mtDNA)

Why is next gen good?

Good for mixed up and degraded ancient DNA, also good for detecting genetic traces of ancient hybridization in modern humans.

What accounts for most of the biodiversity on earth?

Microbiomes

In eDNA samples how can particular species be targeted?

Using taxon-specific primers, followed by qPCR

Why study genetic variation at the molecular level?

1) to determine the genetic basis of inherited disease or phenotypic traits 2) to identify individuals (wildlife ecology) 3) to identify criminals (forensics)

What are Minisatellites?

consists of 10-100 bp sequences that are repeated many time s(up to the thousands) in tandem arrays

Why do Minisatellite arrays (loci) have extremely high allelic variation?

Due to frequent mutations involving replication slippage errors and/or unequal crossing-over

STRs

short tandem repeats

SSRs

simple sequence repeats

What is multiplex analysis?

multiple microsatellites often amplified as the same time, using primers labeled in different florescent colours

How many 'standard’ microsatellite loci are used in criminal forensics? These detect enough variability to distinguish all human individuals (except for identical twins)

T/F. A few microsatellite/STR/SSR loci cause genetic disorders?

True

What happens in the rare cases where microsatellites cause disease?

The loci involve trinucleotide repeats within genes or other important DN sequences.

What can be used to detect DNA polyporphism?

Restriction enzymes

What is the most common cause for restriction site polymorphisms?

Single nucleotide polymorphisms (SNPs)

What is a haplotype?

an arbitrarily long stretch of DNA characterized by particular alleles at the SNP positions in that sequence

Alternative name for microarrays

SNP chips

What is the effect of mutations on restriction endonuclease sites?

can either create or destroy

How can the gain/loss or restriction sites be detected?

Using gel electrophoresis

What is the most common cause for restriction site polymorphisms?

single nucleotide polymorphisms (SNPs)

What is the main goal of crisper (bacteria's original use)?

Defense mechanism against foreign DNA (comparable to adaptive immune system of vertebrates)

CAS

CRISPR ASsociated proteins

Steps for how CRISPR immunity works

1) Spacer Acquisition 2) Expression of crRNAs 3) Interference

What are the three component so interference?

a) Cas9 enzyme b) crRNA c) tracr RNA

Chimeric meaning

combination of different things

What type of cut does Cas9 make in genome?

double-stranded

What is nonhomologous end joining, NHEJ

Broken ends can be rejoined without any template

What is homology directed repair, HDR?

Broken ends can be rejoined using a template

What is the most common type of repair used for double-stranded break in DNA?

NHEJ

What does NHEJ often result in?

insertions and deletions (INDELS)

Main challenges with CRIPSR-Cas9?

1) off-target effects - cleavage sometimes not specific 2) Mosaicism: not all cells edited, different genomes, so get mosaic effect

Main advantage of CRIPSR-Cas9?

Targeting!! Can design single-guide RNA to target (almost) any sequence desired

What is the basic research method? (creating gene knockouts)

Disrupt genes to determine unknown gene functions

T/F. Reversing gene mutations can cause genetic disorders?

True

What do enzymes beta-glucanase, xylanase, and phytase have in common?

They all breakdown matter that pigs donor otherwise digest. Interested for genetic editing to produce them in the pigs salivary glands

When is a 'fetal haemoglobin' expressed?

Before birth

Why are fetal haemoglobin of interest for CRISPR researchers?

Fetal haemoglobin is unaffected by sickle cell anemia and with CRIPSR provides a foundation for a potential 'cure'

What is the most vulnerable group affected by malaria?

Children aged under 5

What can gene drives be used for?

Could be used to insert gene for resistance to malaria, or a gene that reduces fertility of mosquito

What is a gene drive?

a system of biased inheritance in which the ability of a genetic element to pass from a parent to its offspring through sexual reproduction is enhanced

What does gene drive inheritance look like?

Altered gene is ALWAYS inherited

What is pUC 19?

a typical, and commonly used bacterial vector

What does pUC 19 contain?

A portion of the lacZ+ gene with numerous unique restriction enzyme cut sites

What is a unique ER cut site?

sites found nowhere else on the plasmid

ampR

antibiotic resistance gene

Steps to insert foreign DNA sequence into a plasmid?

1) cut foreign DNA with a restriction enzyme 2) cut plasmid with same restriction enzyme 3) mix cut foreign DNA and cut plasmid DNA 4) use DAN ligase to sela sugar-phosphate bonds

What type of plasmid is used to transform competent cells?

Ligated plasmids containing DNA

What is the product of bacteria with non-recombinant plasmid?

beta-galactosidase, resulting in blue colonies

What is the product of bacteria with recombinant plasmid?

(lacZ gene is disrupted by insert) do not produce beta-galactosidase, resulting in white colonies

What allows for expression go genes in bacteria?

included operon and regulatory sequences

What is neomales?

sex-reversed females created by methyl testosterones treatment. All offspring will be females