Genetics quiz 4

In Sanger sequencing, adding a nucleotide without a 3' OH ( a blank) terminates the growing DNA strand.

ddNTP

What best explains why next-generation sequencing platforms yield short reads that must be computationally assembled, while Oxford Nanopore delivers long reads in real time?

nanopore sequences a single DNA molecules as it threads through a pore, reading it continuously end to end, while NGS fragments the library and sequences many copies in parallel- so each read only covers a short fragment

genomic DNA

complete set of genetic instructions an organism carries, the 4 bases arranged along the double helix

total DNA extraction picks up a mixture of…

nuclear and mitochondrial sequences but nuclear genome making up nearly all of the yield

Sequencing a whole genome lets you go beyond just observing phenotype differences between breeds. What does it actually give you?

the genetic forms (variant genes) responsible for the phenotype differences

internal tissue

sit inside a body compartment that environmental genetic material rarely reaches. The risk of bacterial or viral genomes riding along is low, so the extracted DNA accurately represents the host's genome.

Ex of internal tissues

blood, muscle, liver, small ear notch (livestock)

You sequence DNA extracted from an oral swab instead of an ear notch. What does the sequencer read?

a mixture of host and non-host DNA

A project needs the purest possible genomic DNA and can tolerate a more invasive sample. Which choice fits?

ear notch

how much DNA a blood sample actually yields depends on a piece of comparative anatomy which is…

whether the red blood cells have nuclei

erythrocyte

hemoglobin-filled membrane sac — no nucleus, no genomic DNA.

The only DNA in a mammal's bloodstream sits in the blank

leukocytes

ou draw 5 mL of blood from a cow and 5 mL from a chicken, and extract genomic DNA from each. Which sample yields more DNA?

the chicken, its red blood cells are nucleated and carry genomes

Every erythrocyte in a fish, chicken, reptile or amphibian carries

a complete genome

Only the mammalian erythrocyte gives up its blank

nucleus

A mature mammalian red blood cell contains no genomic DNA because it blank its nucleus during maturation in the bone marrow.

extrudes

Frederick Sanger's 1977 insight was that…

you can force DNA copying to stop at predictable places, and the stopping pattern reveals the sequence

ddNTP lacks blank oxygens relative to ribose.

2

ddNTP has no blank for the next nucleotide to attack — once one is incorporated, the chain is dead. It terminates right there.

3’ oxygen

5’ end carries the blank and the 3’ end carried the blank

triphosphate, OH needed for extension

A ddNTP terminates the growing DNA strand because it lacks the blank group on its 3′ carbon.

hydroxyl

In the original Sanger workflow, ddNTPs are supplied at a low concentration relative to dNTPs. What would go wrong if ddNTPs were instead supplied at a high concentration?

chains would terminate too soon, yielding only short fragments

A genomic DNA extraction from an internal tissue comes back with phenol carryover and residual cellular proteins, but the sample is then sequenced anyway. What is the most likely consequence?

the contaminants add noise to the sequencing data, setting a ceiling on the quality of everything downstream

A total DNA extraction from animal cells picks up the nuclear chromosomes, but what other genomic material comes along with it?

small DNA molecules from the mitochondria

A nasal swab was the only feasible sample from a cow. After sequencing, the data includes reads from bacteria, viruses, and food particles alongside host DNA. What must happen before downstream analysis can be trusted?

the non-host must be filtered out computationally before analysis

In a mammal, why does a blood draw yield comparatively little genomic DNA?

only the small white blood cell population carries a genome

In a Sanger reaction, the short blank binds to the template and gives DNA polymerase the 3′ starting point it needs to begin extension

primer

Why does reading a Sanger sequencing ladder require a long polyacrylamide gel?

adjacent fragments differ by exactly one nucleotide in length, and only a long gel can visually separate sizes that close

DNA sequences are always written 5′→3′ because DNA polymerase can only add incoming nucleotides to which end of a growing strand?

the 3’ end, where the hydroxyl group accepts the next nucleotide

lane identity in sanger sequencing tells you…

terminal base

position in sanger sequencing tells you…

fragment length

single-nucleotide resolution:

two fragments differing by exactly one nucleotide must separate visibly.

smaller and faster fragments will end up…

near the bottom

longer and slower fragments will end up…

near the top

On the finished X-ray film, the lowest band — the shortest fragment — corresponds to which base in the sequence?

the first base after the primer

base calling

reading the sequence from the flim

automated base calling

The instrument records the fluorescence signal at high frequency and software converts the color sequence into letters automatically

When software converts the detector's fluorescence color sequence into a file of DNA letters, this is called

automated base calling

AB370

the first commercial DNA sequencing instrument: 16 samples per gel, read lengths of about 450 nucleotides

cycle sequencing

repeated melt–prime–extend cycles dramatically raise the fluorescent signal per reaction.

The AB370 and the Human Genome Project still used Sanger's 1977 chain-termination chemistry. What did automation actually change?

How the method was run and read- fluorescent detection, software base calling, and digital output

Next generation sequencing (NGS)

millions of DNA fragments are sequenced simultaneously on a solid surface, with a camera doing the reading.

NGS platforms are called blank machines

sequencing-by-synthesis

difference between NGS and sanger

instead of one reaction per tube resolved on a gel, millions of fragments are copied in parallel on a flow cell, and each base added is detected optically

A modern benchtop NGS instrument costs roughly $350,000. What does a single run on its flow cell deliver?

about 400 million reads

DNA index

molecular barcode unique to that sample

multiplexing

hundreds of unrelated samples pool into one library and sequence in the same run, later sorted computationally by their barcodes

Because every fragment carries its sample's unique index, hundreds of samples can be pooled into one run — a technique called blank

multiplexing

Sanger and NGS read copies by a blank

polymerase

Oxford Nanopore

reads a single, intact DNA molecule as it threads through a protein pore, with no PCR step and no need to shred the sample into fragments first.