Lecture 16: DNA Sequencing - Genes and Genomes

What is DNA sequencing used for?

DETERMINE nucleotide sequence of unknown DNA stretch (nucleotide by nucleotide)

What is Sanger (dideoxy) sequencing, and what does it rely on?

• DNA sequencing method

• USES dideoxynucleotides (ddNTPs)

What are the four types of ddNTPs?

• ddATP

• ddTTP

• ddGTP

• ddCTP

What is included in the Sanger sequencing reaction mix?

• Template DNA

• Primer

• DNA polymerase

• dNTPs

• Labeled ddNTPs

How do dNTPs and ddNTPs differ in DNA synthesis?

• dNTPS → have 3’ -OH group (allows elongation)

• ddNTPs → lack 3’ -OH (terminates elongation)

Why do dideoxynucleotides terminate DNA strand elongation?

WITHOUT 3′ –OH group, DNA polymerase cannot add another nucleotide

How are dNTPs and ddNTPs labeled in Sanger sequencing?

• dNTPs → unlabeled

• ddNTP → each type labeled with different fluorescent dye

How do dNTPs and ddNTPs differ in DNA synthesis?

• dNTPs → allow elongation

• ddNTPs → terminate elongation

How is the primer allowed to anneal (bind) in Sanger sequencing?

Heat and cool the template DNA and primer once

What happens after the primer anneals?

• DNA polymerase is added

• ONE round of DNA synthesis occurs

What happens when ddNTPs are added to a growing DNA strand?

DNA synthesis terminates (because lacks 3’ –OH group)

What happens during each step of Sanger sequencing?

• SOME DNA strands terminate (ddNTPs added)

• OTHER strands continue elongating (dNTPs added)

What is the end result of a Sanger sequencing reaction?

• DNA fragments of different lengths

• EACH fragment labeled with a fluorescent dye

Why do DNA fragments have different lengths after Sanger sequencing?

DIFFERENT strands terminate when labeled ddNTP is incorporated

How are sequencing products separated?

DNA fragment size

How is the DNA sequence determined from Sanger sequencing results?

• ANALYZE fluorescent signal of DNA fragments

• ORDERED by fragment length

• TO DEDUCE (conclude) the sequence

How many base pairs can one Sanger sequencing reaction read?

FEW hundred to thousands of base pairs

What are the three main steps of shotgun sequencing?

1. CUT genome into small fragments (via restriction enzymes)

2. SEQUENCE each fragment

3. ASSEMBLE sequence

What is shotgun sequencing used for?

• SEQUENCING entire genome

• READS and ASSEMBLES many small DNA fragments

What is Illumina sequencing?

METHOD sequencing millions of DNA fragments simultaneously on a microscope slide

What does each spot on an Illumina sequencing slide represent?

DNA fragment being sequenced

What type of nucleotides does Illumina sequencing use?

REVERSIBLY terminating nucleotides

What are reversibly terminating nucleotides?

• MODIFIED DNA building blocks

• TEMPORARILY pause DNA synthesis during sequence

How do reversibly terminating nucleotides work?

• CHEMICAL block temporarily prevents elongation

• REMOVING allows synthesis to continue

What happens during each cycle of Illumina sequencing?

• ONE nucleotide added and detected

• TERMINATING chemical group removed

How is a whole genome assembled from Illumina sequencing results?

• USE overlapping sequences to assemble fragments

• INTO continuous genome sequence

What is the key difference between Sanger and Illumina sequencing?

• Sanger sequences → individual DNA fragments

• Illumina sequences → millions simultaneously

What is the role of restriction enzymes in DNA sequencing?

• CUT DNA at specific sequences

• PRODUCES fragments that can be sequenced

How is the E. coli genome organized and why?

• ONE circular double-stranded DNA molecule

• BECAUSE it’s bacteria

How is bacterial DNA organized in the nucleoid?

• LOOPS of supercoiled (winded) DNA

• ATTACHED to protein core

How is the human genome organized?

• MULTIPLE linear chromosomes

• EACH containing one double-stranded DNA

What do DNA-binding proteins do in eukaryotes?

PACKAGE DNA into chromatin

How does bacterial DNA organization differ from eukaryotic DNA organization?

• Bacteria → ONE circular chromosome

• Eukaryotes → MULTIPLE linear chromosomes (chromatin)

What is a nucleosome?

DNA wrapped around histone proteins

What are the four core histone proteins?

• H2A

• H2B

• H3

• H4

What are the charges of DNA and histone proteins?

• DNA → negatively charged

• Histones → positively charged

What is the 10-nm and 30-nm chromatin fiber?

• 10-nm → NUCLEOSOME beads on DNA string

• 30-nm → COILED nucleosome fiber

What is genome annotation?

IDENTIFYING genomic features (e.g., genes and promoters) and their locations

What methods are used in genome annotation?

• Computational

• Mathematical

• Statistical

What is a reading frame?

METHOD of grouping DNA/RNA sequence into triplet codons

How many reading frames does double-stranded DNA have?

Six (three per strand)

What is an open reading frame (ORF)?

• SEQUENCE

• STARTS with start codon

• CONTINUES to stop codon

How do researchers identify protein-coding genes in bacterial genomes?

• COMPUTER programs search for ORFs

• CONSERVED promoter sequences help to identify

Why is identifying protein-coding genes harder in eukaryotes?

EUKARYOTIC genes contain introns interrupting coding sequences

How can cDNA help identify eukaryotic genes and why?

• USE cDNA (complementary DNA) sequences to locate corresponding genes in genome

• INTRONS removed

What is the G-value paradox?

NUMBER of (coding) genes in organism not good predictor of biological complexity

How can one gene produce multiple proteins?

• ALTERNATIVE splicing produces different mRNAs from one RNA transcript

• LEADS to different proteins

How can one protein have multiple functions?

• COMBINING proteins into different complexes

• POST-TRANSLATIONAL modification of proteins (to alter function)

What is the C-value paradox?

NO relationship between genome size and biological complexity

What is the difference between a gene and a genome?

• Gene → DNA sequence producing functional product

• Genome → organism’s complete set of DNA

What are two explanations for why there are differences in genome size?

• POLYPLOIDY → having more copies of the same genes (multiple chromosome sets)

• NONCODING DNA → not all DNA codes for proteins

What percentage of the human genome codes for proteins?

2.5%

What percentage of prokaryotic DNA typically codes for a product?

Approximately 90%

What are transposable elements?

• PARASITIC segments that can move from one location in genome to another

• PRIORITIZE own replication and transmission (within genome)

What are short tandem repeats (STRs)?

DNA sequences repeated consecutively

What are microsatellites (simple sequence repeats)?

REPEATING sequences of 1–5 bases

What are minisatellites (variable number terminal repeats [VNTRs])?

REPEATING sequences of 6–500 bases

Why are repeat sequences called hypervariable?

• REPEAT numbers and lengths (how many times DNA sequence repeats) vary among individuals

• EACH individual has unique set

Why are STRs useful in DNA fingerprinting?

REPEAT lengths vary among individuals (helps identify)