W - BIOL200 - 3.9-3.12, GENOMIC MAPPING, WHOLE GENOME SEQUENCING, NUCLEIC ACID DETECTION

What is gene mapping?

Finding the positions (locations) of genes on chromosomes.

What is linkage analysis?

 Determines which genes are close together (linked) on a chromosome.

 How can different alleles be identified?

Using SNPs or RFLPs.

 What are SNPs?

 Single nucleotide polymorphisms—single base pair changes in a gene.

What are RFLPs?

Differences in DNA fragment lengths after digestion with restriction enzymes due to SNPs at restriction sites.

What effect does an SNP at a restriction site have on RFLPs?

 Prevents cutting → different band patterns on gel electrophoresis.

 What are the 5 main steps in DNA sequencing?

 Purification, Fragmentation, Amplification, Sequencing, Reassembly.

What is Sanger sequencing?

Uses ddNTPs to terminate DNA synthesis and fluorescent tags to identify bases.

How does Sanger sequencing stop replication?

ddNTPs lack 3' OH, terminating elongation when incorporated.

How are Sanger sequencing products separated?

Electrophoresis (based on size).

What does each fluorescent tag in Sanger sequencing indicate?

A specific nucleotide base (A, T, G, C).

What does fragment length tell you in Sanger sequencing?

 Shortest to longest = base order.

What is whole genome shotgun sequencing?

Breaks DNA into fragments, sequences them, and aligns overlaps with a computer.

What is next-generation sequencing (NGS)?

Uses one instrument to do billions of reactions simultaneously.

How does NGS work?

DNA is linked, amplified, bonded to a solid surface, and sequenced by detecting fluorescent bases.

What does a Southern blot detect?

Specific DNA sequences.

What does a Northern blot detect?

Specific RNA sequences.

What is the common step in both blot types?

Gel electrophoresis followed by transfer to membrane and hybridization with labeled probes.

What labels are used in probes?

Fluorescent or radioactive tags.

What allows hybridization to occur in blots?

Complementary base pairing between probe and target.

 What is measured in a Northern blot?

Specific mRNA levels using radiolabeled probes.

What are the steps of gene cloning?

 Amplify gene → insert into plasmid → transform bacteria → express gene → purify protein.

What are restriction sites?

DNA sequences recognized and cut by restriction enzymes.

What are sticky ends?

Overhanging DNA sequences that allow ligation.

What enzyme joins sticky ends?

DNA ligase.

What must primers contain to amplify a gene for cloning?

Sequences matching restriction sites.

What is an Ori in a plasmid?

Origin of replication.

What is an antibiotic resistance gene in a plasmid used for?

Selection of transformed bacteria.

What are restriction enzymes?

Enzymes that cut DNA at specific sequences (restriction sites).

Where do restriction enzymes originate from?

Bacteria—used to cut viral DNA.

What types of ends can restriction enzymes create?

Sticky ends or blunt ends.

What is an example of a palindromic restriction site?

5'...CTCGAG...3' (cut by XhoI)

What is required for inserting a gene into a plasmid?

 Restriction sites at gene ends and matching sites in the plasmid.