Lecture 16 - DNA Sequencing Genes and Genomes

DNA sequencing

• Sequencing is used to determine the nucleotide sequence of an unknown stretch of DNA, nucleotide by nucleotide

Sanger Sequencing

• most widely used method of DNA sequencing

• relies on dideoxynucleotides or ddNTPs, i.e, ddATP, ddTTP, ddGTP, and ddCTP

• reaction mix: template DNA, primer, DNA polymerase, dNTPs, labelled dideoxynucleotides (ddNTPs)

Deoxynucleotides vs Dideoxynucleotides

• A deoxynucleotide has a hydroxyl group on the 3’ carbon, allowing this end to be elongated

• A dideoxynucleotides lacks the 3’ hydroxyl group, and it cannot be elongated by DNA polymerase (each nucleotide is labeled with different fluorescent dye)

Dideoxy DNA sequencing

• Similar to PCR, we heat and cool the reaction mix with template and the primer to allow the primer to anneal (just once though)

• After that, DNA polymerase is added and a SINGLE round of DNA synthesis takes place

Dideoxy DNA sequencing Steps

• In each subsequent step, synthesis will be terminated on some strands (ddNTP is added), and synthesis will be continued on others (dNTP is added)

Dideoxy DNA Sequencing end result

• synthesis of DNA chains with a range of sizes, each labeled with a fluorescent dye

Products of a DNA sequencing reaction

• Since the dNTPs do not have a label, we cannot see them

• We can see the ddNTPs though

How are the sequency products separated during Dideoxy DNA sequencing?

• by size

• insert graph later

Sequencing whole genomes

• A single sequence reaction will yield a few hundred to a few thousand bp (base pairs) of sequence

Shotgun Sequencing

1. cut genome into small fragments (use of restriction enzymes)

2. sequence each fragment (illumina sequencing)

3. assemble sequence

Illumina sequencing

• allows us to sequence millions of DNA fragments on a microscopic slide

• uses reversibly terminating nucleotides in the sequencing reaction

• with the terminating chemical block, it cannot be elongated

• once the chemical block is removed, it can be elongated

How are the sequences assembled during shotgun sequencing?`

• sequence overlaps to assemble whole genome sequence — requires heavy computing!

What are genes and genomes?

Gene: a specific segment of DNA that provides instructions to make proteins

Genome:the complete set of genetic material or DNA in an organism

Nucleoid

loops of supercoiled DNA attached to a protein core

Genome organization in eukaryotes

DNA binding proteins package DNA into chromatin

Genome annotation

• identifying various sequence features (genes, promoters, etc) and their locations in the genome

• requires computational, mathematical and statistical methods

How do we find protein coding genes?

• computer programs are used to search a genome sequence to identify open reading frames (ORFs)

What is a reading frame?

a way of dividing a sequence of nucleotides (DNA or RNA) into consecutive codons

What is an ORF

ORF = AUG - (codon-codon-codon)_n-STOP

n = number of codons in the protein (a few dozen to several hundred)

Bacteria and Archaea

• no introns

• genes can be identified by highly conserved promoter sequences associated with a distinct ORF

EU

Eukaryotic Organisms

• genes contain introns

• identification of ORFs is more difficult

• One strategy is to use cDNA sequences and then locate the gene in the genome sequence

G-value paradox

• the number of genes in an organism is not a good predictor of biological complexity

• Explanation: different proteins can be made from a single gene, depending upon how the RNA transcript is spliced together

• Another explanation: more functions for each protein

C-value paradox

• no relationship between genome size and biological complexity

• explanation #1: polyploidy - having more copies of the same gene

• explanation #2: noncoding DNA - not al DNA codes for protein

Genes vs Genomes

• Protein-coding sequences constitute about 2.5% of the human genome, and repeated sequences make up more than 50%

• in prokaryotic cells, usually 90% of the DNA sequences code for a product used by the cell

Repetitive Sequences in Eukaryotes

1. transposable elements (~45% of the genome)

2. short tandem repeats (~3% of the genome)

Transposable Elements

• parasitic segments of DNA able to move from one location in a genome to another

Short Tandem Repeats

• microsatellites, or simple sequence repeats, are repeating sequences of 1 - 5 bases

• minisatellites, or variable number terminal repeats (VNTRs), are repeating sequences of 6 - 500 bases

• Repeat sequences vary among individuals, each individual has a unique set

• Repeat sequences can be used in DNA fingerprinting by analyzing repeat length

Humans and Chimpanzees

• The base sequences of human beings and chimpanzees are 98.8% identical

• the leading hypothesis focuses on the importance of regulation of gene expression