Lecture 18: Genomics and Sequencing

genome

The _______ is all the genetic information that defines an organism.

Various processes

________ are encoded by the genome. Ex. Energy and nutrient sources (catabolism), growth products (biosynthesis), factors that promote fitness (ecology), molecular adaptions (evolution).

Genomics

science of mapping, sequencing, analyzing and comparing genomes between species.

Haemophilus influenzae

The first bacterial genome that was sequenced was ________________ in 1955.

Open reading frame (ORF)

A region of DNA that begins with the start codon and ends with a stop codon.

more complex, greater

Generally the _________ an organism the ______ the genome size.

Sequence, Assemble, Annotate

Genome of an organism can be derived using the following steps

1. Sequence

Determining the order of the bases within DNA

2. Assemble

Piece together different parts of the genome in the correct order.

3. Annotate

Convert this raw data into a list of genes whose function may or may not be known.

Bioinformatics

is the process of using a computer to store and analyze the sequences of structures of DNA and proteins

functions

Once sequenced a genome can provide insight into how an organism ________.

DNA synthesis

sanger sequencing designed by Fred Sanger this technique uses __________ to determine the sequence of a region of DNA>

replicating, amplifying

Sanger sequencing involves ________ and ________ a strand of DNA using DNA polymerase. This is done via a similar method to PCR.

terminated, fluorescently labelled, ddNTPs (dideoxyribonucleotides)

At random points DNA replication is ___________, leaving a _____________ residue at the end. This is done using fluorescently labeled _____________ of all four bases in the reaction.

specific, sequence

Each base has a __________ fluorescent label of a different color. This allows us to determine the ________ of the DNA.

Dideoxyribose-NTPs (ddNTPs)

can be integrated into the DNA and differ from dNTPs in two ways

They lack a 3' OH, terminal nucleotides

This means there is no way to extend the chain past the ddNTP, as further monomers cannot be added. They are referred to as _______________.

random, very single

This integration is _______, with millions of DNA strands made during sequencing it is inevitable that a ddNTP will be present in ___________ residue

base

Each ddNTP has a different fluorescent which enables us to determine which terminal ________ is present at the each of each strand of DNA created.

DNA replication mixture (Step 1 of Sanger sequencing)

Fluorescently labeled ddNTPS and regular dNTPs are added to a ____________, with the target strand of interest and a known primer.

replication stops, ddNTP, every position (Step 2 of Sanger sequencing)

DNA synthesis continues until a ddNTP is integrated. At this point ___________. There will be a ______ integrated at _________ of the DNA.

capillary electrophoresis (Step 3 of Sanger sequencing)

The fragments are separated by size using ____________, the shorter the fragment the faster it will migrate down the column.

assembled (Step 4 of Sanger Sequencing)

Automated sequencer reads outputs, inputting the corresponding base, DNA sequence can be __________.

smallest, further

Remember, the _________ fragment will always migrate ________.

molecular sieve

Smaller fragments migrate further because the column acts as a ____________ therefore it takes larger samples longer to get through the pores.

smaller, earlier, reverse complementary

The ____ the fragment the ______ it comes in sequence. The sequence __________ to the sequence we determine as synthesis is 5' to 3'.

primer, known sequence

Sanger sequencing requires the use of a _______. This must bind to a __________.

800, known, large-scale

There are several limitations to Sanger sequencing. Limited to around _______ nucleotide per reaction. Can only be sequence from _________ sequence. Cannot be used in _________ genome sequencing.

fragment the DNA, numerous reactions

To sequence an entire genome or a larger region of DNA you would have to first _________ into multiple, smaller pieces. ______________ would be required to completely sequence the genome.

assembled

Once a genome has been sequenced it must be ________ before it can be analyzed.

correct order, overlaps

Assembly entails putting the fragments in the _______ to reconstruct the chromosome and eliminating any _________.

looks for overlaps

To assemble a genome the computer analyzes the sequences produced by next-generation sequencing and _____________.

jigsaw puzzle, consensus genome

Like a _________ the overlaps indicate that pieces should fit next to one another. This produces a ___________.

annotated

Once the genome has been assembled it can be _________.

Gene annotation

the identification of genes and other functional regions within the DNA

open-reading frames, start codon.

The annotation relies on finding __________, which begin at a _____________.

protein sequence, stop codon

Once a start codon is found in annotation the software will look for a potential ________ ending at a _________.

ribosome binding site

The software will then determine whether there is a canonical ______________. (for annotation)

valid or not

The software will predict whether the found ORF is _________ based on a database (annotation).

translated

If an ORF is determined to be valid the DNA sequence is ____________. (annotation)

protein functional domains

The protein sequence is compared to that of known __________________. (annotation)

share a functional domain, common, similar

Proteins which ______________ often have a _________ evolutionary ancestor that may share a ___________ role. (annotation)

BLAST (basic local alignment search tool)

Many bioinformatic tools are available to help annotate a protein correctly, such as GenBank's ________.

protein sequence

When attempting to determine the function of a ORF the _________ is used instead of the DNA sequence.

redundancy, identical

We use the protein sequence instead of the DNA sequence when determining the ORF because codons have _________, several codons encode the same amino acid. Therefore some genes may not have a similar sequence, but protein sequence may be __________.

different

Different organisms have ________ codon usage.

horizontally transferred

The difference in codon frequency usage can indicate which genes have been _____________. (ex. An E. coli gene which encodes purely AGG arginine codons may have come from another bacteria.)