exam 1

cut nucleotides

restriction enzymes

piece of DNA or RNA that has been amplified through PCR

amplicon

where are restriction enzymes found

bacteria

enzymes that degrade DNA or RNA

nucleases

identical DNA/RNA strands bind to each other, 2 separate molecules

dimer

2 primers bind to each other forming an amplicon

primer dimer

protects cellular DNA by marking native DNA with methyl group

modification enzyme

enzyme that cuts or degrades DNA

deoxyribonuclease

enzyme that cleaves nucleic acid molecules in the middle

endonuclease

enzyme that cleaves nucleic acid molecules at the end, removes a single nucleotide

exonuclease

sequence of DNA bases that are recognized by a specific protein (ex. restriction enzymes)

recognition site

enzyme that cuts or degrades RNA

ribonuclease

cuts at a location far from recognition site

Type 1

cuts in middle of recognition site, position of cut location is known

Type 2

You are working with a circular plasmid and want to insert a gene of
interest using a restriction enzyme. You have two enzymes to choose
from: EcoRI and HindIII. EcoRI cuts the plasmid once and HindIII cuts
it twice. Which enzyme would you choose to create a recombinant plasmid
with your gene insert?

EcoRI

breaking open cells to by disrupting cell membranes and nuclear envelopes to release DNA, protects it from degradation, inactivates DNases

DNA lysis

Detergents, salts, chaotropic agents, buffering agents, enzymes

types of DNA lysis chemicals

chaotropic salts disrupt water structure and promote binding of DNA to silica

DNA binding

DNA precipitation

uses UV absorbance at 260nm to measure DNA or RNA concentration

nanodrop

Which of the following is a primary function of an extraction
buffer in DNA isolation?

To break open cells and protect DNA from degradation

Which statement best compares the NanoDrop and Qubit methods of DNA quantification?

Nanodrop provides purity ratios, while Qubit offers more accurate DNA quantification

Amplifies a small amount of DNA

PCR

Order the following PCR steps:

1. Annealing

2. Extension

3. Denaturation

312

primer, short sequence designed ahead of time

oligonucleotide

enzyme responsible for synthesizing DNA during DNA replication

DNA polymerase

enzyme in some RNA viruses that converts RNA into DNA, integrates DNA into host genome

reverse transcriptase

Tracks DNA replication in real time using fluorescence

qPCR

Which is useful for making copies of a gene of interest

TA

Which is formed from a primer whos ends are complementary?

hairpin

barcoding helps you ID unknown species

true

which is sequencing by synthesis

illumina

which of the following refers to the fluorescently labeled nucleotides used in sequencing

ddNTP

Put the steps of TA cloning in order:

1. fragment genome

2. select successful clone with antibiotics

3. transformation

4. isolate gene of interest

4132

what is the range of a p1000 pipette?

100-1000ul

which is useful in determining the quality of a sequence read?

chromatogram

Which can be used to compare an unknown sequence to a known sequence database?

basic local alignment search tool

Which refers to DNA produced from mRNA template?

cDNA

Which is not a PCR ingredient?

endonuclease

Which is used for visualizing PCR products?

gel electrophoresis

TOPO TA cloning utilizes

topoisomerase

clusters similar sequences together

OTU

identifies individual sequences while removing errors

ASV

best for pure culture ID, plasmids, or detecting deletions

sanger

uses ddNTPS that stop DNA synthesis and separates fragments by size and detects fluorescent tags, producing a chromatogram

sanger

best for sequencing microbiomes and reconstructing genomes

illumina

incorporates fluorescent bases to sequence by synthesis

ilumina

best for de novo sequencing and genome reconstruction

nanopore

dna goes through a pore, which causes voltage changes that can be read as different bases

nanopore

cheaper and portable option

nanopore

most versatile method

ilumina

sequences up to 800 bp

sanger

sequences 300-500 bp

illumina

no size limit to sequencing

nanopore

cheap, but need a very pure sample

sanger

does a forward and reverse read

illumina

Order the steps of cloning

1. ligation

2. selection of clones and downstream procedures

3. transformation

4. isolation of DNA of interest

4132

cuts gene of interest and vector with same enzyme, results in overhangs of complimentary single strand

restriction enzyme cloning

using PCR to make cut and insert PCR product into vector

TA cloning

Taq adds one A to PCR product, product mixed with plasmid vector with T overhang and topoisomerase I join vector and insert

TOPO TA cloning

Taq adds one A to PCR product, product mixed with plasmid vector with T overhang and ligase to join vector and insert

TA cloning

artificially engineered versions of plasmids that are improved with antibiotic resistance

vectors

process through which our construct will be taken up by competent bacterial cell

transformation

only cells that incorporate vector and have antibiotic resistance genes survive

screening

why is cloning used

approaches to purify nucleic acid

Order the steps of DNA extraction

1. remove membrane lipids with detergent

2. washing to remove impurities

3. separate cells from matrix

4. remove proteins through protease

5. break cells open (lysis)

6. precipitation or binding of DNA

351462

protects dna from degradation, disrupts membranes, inactivates nucleases

DNA lysis

detergents, salts, chaotropic agents, and buffering enzymes are used to

lyse DNA

phenols, silica columns, and magnetic beads are used in

DNA lysis

chaotropic salts disrupt water structure to promote binding of DNA to silica via hydrogen bonding and salt bridges

DNA binding

uses ethanol or isopropanol to precipitate nucleic acids, forms a DNA pellet

DNA precipitation

absorbance based method of assessing DNA concentrations

nanodrop

uses UV absorbance at 260nm to measure DNA or RNA concentration

nanodrop

260/280

protein contamination

260/230

salt/solvent contamination

uses fluorescent dyes that bind to DNA, DNA, or proteins to assess DNA concentrations

qubit

Which of the following best describes the purpose of cloning a gene into a
plasmid vector?

to amplify and manipulate the gene in a stable, replicable system

identifies a species using a short standardized region of DNA that is unique to each species

barcode

widely used barcode for bacterial identification, encodes ribosomal subunit in prokaryotes

16S gene

identifies multiple species from mixed samples using high throughput sequencing

metabarcoding

sanger sequencing, uses chain terminating nucleotides and electrophoresis to sequence, shorter read lengths

early sequencing

illumina, parellel sequencing millions of molecules simultaneously, requires amplification

next generation sequencing

nanopore, sequences individual DNA molecules directly, no PCR required, reads 10kb-1mb

third generation sequencing

uses for third generation sequencing

de novo sequencing, resolving repetitive regions

uses for next generation sequencing

medicine and biology

uses for early sequencing

targeted sequencing and validation