Genetics Module 9

Why do scientists break a genome into small DNA fragments before sequencing it?

A genome is too large to sequence as one piece. Scientists sequence smaller fragments and use overlapping DNA sequences to reconstruct the original genome.

What is the overall workflow for analyzing a genome?

Fragment the DNA → clone/amplify fragments → determine their sequences → use overlaps to assemble the genome. The slides introduce annotation as the next step after sequencing.

Why did bacteria evolve restriction enzymes?

To defend against foreign DNA, such as bacteriophage DNA. Restriction enzymes recognize certain sequences and cut the invading DNA.

How can methylation help protect DNA from restriction enzymes?

Methylation at appropriate recognition sites can prevent restriction enzymes from cutting the DNA. This helps explain restriction versus protection of phage DNA in the lecture.

What exactly does a restriction enzyme recognize?

A specific DNA sequence, commonly 4–6 base pairs long and often palindromic, where the enzyme makes a double-strand cut.

What does 'palindromic recognition site' mean in DNA?

The sequence reads the same 5′→3′ on both complementary strands. Example: 5′-GAATTC-3′ paired with 3′-CTTAAG-5′.

How can you tell if a restriction enzyme makes blunt ends?

The two strands are cut directly opposite one another, so neither fragment has a single-stranded overhang.

How can you tell a 5′ sticky end from a 3′ sticky end?

Look at which end of the single-stranded overhang is exposed: an overhang ending in a free 5′ end is a 5′ overhang; one ending in a free 3′ end is a 3′ overhang.

Why are sticky ends helpful for cloning DNA?

Complementary overhangs can base-pair with one another, holding an insert next to a cut plasmid so DNA ligase can seal the backbone.

Can any two restriction enzymes that make sticky ends join their fragments?

No. The overhang sequences and their orientations must be compatible. Both enzymes making sticky ends does not automatically make their products compatible.

If an enzyme recognizes a 6-base sequence, how far apart are its sites on average in random, equal-base DNA?

4^6 = 4,096 bp on average. The rule assumes A, T, G, and C each occur 25% of the time and are randomly distributed.

Why do 8-base cutters usually produce longer DNA fragments than 4-base cutters?

A specific 8-base sequence is rarer than a specific 4-base sequence. Fewer cuts means larger average fragments: 4^8 = 65,536 bp versus 4^4 = 256 bp.

Practice: A 5-base recognition site in equal-base DNA gives what average fragment size?

4^5 = 1,024 bp. Count the bases in the recognition site, then raise 4 to that number.

Practice: A genome is 12,800,000 bp and an enzyme cuts every 256 bp on average. How many fragments?

12,800,000 ÷ 256 = 50,000 fragments, approximately. Divide genome length by average fragment length.

Practice: A hypothetical organism has six equally likely bases and an enzyme recognizes 4 bases. What changes?

Use 6^4, not 4^4: the average fragment is 1,296 bp. The base of the exponent is the number of equally likely nucleotide types.

Why can't you always use 4^n to estimate restriction fragments?

It assumes equal frequencies of A, T, G, and C. If frequencies differ, find the probability of the exact recognition sequence by multiplying the probabilities for each position.

Practice: If G = 40% in double-stranded DNA, what are C, A, and T?

C = 40%, A = 10%, T = 10%. Chargaff's rules give G = C and A = T, with all four totaling 100%.

Practice: With A=T=10% and G=C=40%, how often is ClaI's ATCGAT site expected?

P(ATCGAT) = (0.1)(0.1)(0.4)(0.4)(0.1)(0.1) = 0.000016, or once per 62,500 bp on average.

Practice: For that ClaI site, about how many fragments form from 3 billion bp?

3,000,000,000 × 0.000016 = 48,000 fragments, approximately. Equivalently, divide by 62,500 bp per fragment.

Why does DNA move toward the positive electrode in gel electrophoresis?

DNA's phosphate backbone is negatively charged, so an electric field pulls it toward the positive electrode.

On a DNA gel, which travels farther: 200 bp or 2,000 bp, and why?

200 bp travels farther because small DNA fragments move through the gel matrix more easily. Small = farther; large = closer to wells.

If two DNA bands are at the same height on a gel, what can you infer?

They contain fragments of approximately the same length, not necessarily the same DNA sequence.

What's the point of cloning a DNA fragment into a plasmid?

The plasmid carries the DNA fragment into bacteria; as bacteria grow and replicate their plasmids, many copies of that fragment are produced.

What does a polylinker (multiple cloning site) contribute to a plasmid?

It gives several restriction enzyme recognition sites where an inserted DNA fragment can be placed.

Why must a cloning plasmid have an origin of replication?

It allows the plasmid DNA to be copied in the intended host. Without a functioning origin, the plasmid cannot be maintained through replication.

Why does a cloning plasmid need a selectable marker?

It distinguishes cells that received the plasmid from those that did not. An antibiotic-resistance gene is a common example.

What is the difference between restriction enzymes and DNA ligase during cloning?

Restriction enzymes cut DNA at specified sites; DNA ligase joins DNA pieces by sealing the sugar-phosphate backbone.

Put these cloning steps in order: select, ligate, cut, transform.

Cut the plasmid and insert → ligate them → transform bacteria with the DNA → select bacteria that took up the plasmid.

What does 'transformation' mean in plasmid cloning?

Bacterial cells take up plasmid DNA from outside the cell, often after treatments that temporarily make their membranes more permeable.

Why plate transformed bacteria on ampicillin?

The ampicillin-resistance marker lets bacteria carrying the plasmid survive while bacteria without it generally cannot grow.

A colony survives ampicillin. Does that prove it has the correct gene insert?

No. It indicates the bacterium carries the resistance marker, but the plasmid could lack the intended insert. Verify by digestion, gel analysis, or sequencing.

What is a genomic library, in plain language?

A collection of cloned DNA fragments that together represent the genome, like books that collectively cover an entire text.

Why does the size of the restriction enzyme recognition site affect genomic library size?

More frequent cutting produces more, smaller inserts and therefore more different clones needed to cover the genome (assuming suitable vectors and complete coverage).

What is the drawback of completely digesting DNA with a single restriction enzyme?

Every site gets cut, producing adjacent fragments that do not overlap. Without overlap, figuring out their original order is harder.

How does partial digestion make genome assembly easier?

It cuts only some recognition sites, generating DNA fragments of different sizes that overlap. Matching overlaps helps place fragments in order.

How can scientists make a restriction digest partial rather than complete?

Decrease restriction enzyme concentration or shorten reaction time, as the lecture specifies.

How is mechanical shearing different from restriction digestion?

Mechanical shearing physically breaks DNA without relying on enzyme recognition sequences; it can generate overlapping fragments for reassembly.

What does 'contig' mean?

A continuous reconstructed DNA sequence assembled by aligning overlapping shorter reads or fragments.

Practice: Read 1 = ATGCC; Read 2 = GCCTAA. What is the assembled sequence?

ATGCCTAA. The end 'GCC' of Read 1 overlaps the beginning 'GCC' of Read 2, so write the overlap only once.

What components are essential for Sanger sequencing?

Template DNA, primer, DNA polymerase, normal dNTPs, and fluorescent chain-terminating ddNTPs; fragment separation and fluorescence detection reveal the sequence.

Why do you heat the DNA before starting Sanger sequencing?

Heat separates the two strands so a primer can anneal to the template and DNA polymerase can start extending it.

What is the primer's job in Sanger sequencing?

It binds the template and supplies the free 3′-OH from which DNA polymerase can begin DNA synthesis.

What is the key difference between a dNTP and a ddNTP?

A normal dNTP has a 3′-OH that supports continued synthesis; a ddNTP lacks that 3′-OH, so incorporation ends the growing chain.

Why do Sanger reactions produce fragments of many different lengths?

Normal dNTPs extend DNA, but occasional incorporation of a fluorescent ddNTP stops a strand at a particular position.

What does the fluorescent color on a Sanger fragment tell you?

Which base (A, T, G, or C) was the chain-terminating ddNTP at the end of that fragment.

Why are Sanger fragments separated by electrophoresis?

Their lengths reveal the order in which bases were added. The shortest terminated products are read first, then progressively longer ones.

When reading Sanger fluorescence peaks from shortest to longest, whose sequence do you get?

The newly synthesized strand in the 5′→3′ direction, not the template strand.

Practice: Sanger reads new strand 5′-ACGT-3′. Write the template strand aligned to it.

3′-TGCA-5′. The template is complementary and antiparallel to the new strand.

What happens if DNA polymerase is omitted from a Sanger reaction?

No new DNA is synthesized, so there is no normal ladder of terminated sequencing products.

What happens if ddATP is omitted from a Sanger reaction?

Fragments cannot terminate through incorporation of ddATP, so the expected A-terminated labeled products are absent.

What happens if normal dCTP is omitted from a Sanger reaction?

The polymerase cannot normally extend through positions needing C; ddCTP can terminate at such positions, preventing continued extension there.

How does whole-genome shotgun sequencing reconstruct a genome?

Create a library of many short DNA fragments (the slides say 500–1,000 bp), sequence random fragments, and use shared overlaps to assemble contigs.

Why do repetitive DNA regions cause problems in shotgun assembly?

Reads from identical repeats can match multiple locations, so the assembler may connect pieces in the wrong order or location.

How does paired-end sequencing provide more information than reading just one end?

It sequences both ends of the same larger DNA fragment, linking the reads with a known approximate distance and orientation to help span repeats.

Practice: Two matching repeat reads could belong to different genomic locations. What information helps decide where they go?

Paired-end reads whose mates map to flanking unique sequences, together with their expected separation, help place the repeat correctly.

Final check: What's the core difference among cloning, Sanger sequencing, and shotgun sequencing?

Cloning makes many copies of an inserted fragment; Sanger determines the nucleotide order in a fragment; shotgun sequencing reconstructs a genome from many randomly sequenced, overlapping fragments.