Chapter 2: 2.1 Sequencing Flashcards
What are the key steps to undergo if you want to sequence a genome?
- Purification
- Fragmentation
- Amplification
- Sequence fragments
- Re-assembly of fragments
Define:
Purification
Isolating DNA from an organism or cells
Define:
Fragmentation
Breaking up the genome into smaller fragments to be sequenced
Define:
Amplification
Make more copies of our fragments
Define:
Sequence fragments
Assigning nucleotide bases to our fragments
Define:
Re-assembly of fragments
Put all the sequence fragments back together to create a continuous sequence
List:
Types of Sequencing
- Sanger Sequencing
- Whole Genome Shotgun Sequencing
- Next Generation Sequencing
What is the Sanger Sequencing Method also known as?
The dideoxy method
Describe:
Dideoxynucleotides
Have an H on the 3’ carbon of the sugar-phosphate backbone instead of OH attached to a fluorescent tag
How does Sanger Sequencing work? (5)
- Amplified fragments are replicated again in the presence of the ddNTPs
- Replication enzyme uses normal nucleotide bases and then randomly inserts a ddNTP that stops replication
- Fluorescently labeled sequencing fragments are run through electrophoresis to separate them by size
- The fluorescent tags are all different colors for their respective nucleotide base, need to subject gel to fluorescent filter
What is the order of sequence in Sanger Sequencing?
Shortest to longest (fragment that travelled most to fragment that travelled least)
What is Sanger Sequencing most useful in?
Most useful in sequencing single genes
What is Whole Genome Shotgun Sequencing most useful in sequencing?
Useful in sequencing the entire genome
How does Whole Genome Shotgun Sequencing?
- Isolate genome DNA and break up into overlapping fragments
- Clone each fragment into a plasmid vector
- Sequence the genomic DNA fragment in each clone
- Use computer programs to re-align sequences based on areas of overlapping
In Whole Genome Shotgun Sequencing:
How is genome DNA isolated and broken up into overlapping fragments?
By using 2 DNA samples that have been digested by 2 different restriction enzymes
What does Next Generation Sequencing allow for?
Allows one sequencing instrument to carry out billions of sequencing reactions at the same time
What is Next Generation Sequencing useful in sequencing?
Useful for multiple gene sequencing of whole genome sequencing
How does Next Generation Sequencing work?
- DNA fragments prepared by adding double stranded linkers to both ends of the fragment
- Fragments are amplified using PCR primers that are complementary to linkers
- Primers are then used to covalently bond the DNA fragments to a solid surface in a tight cluster
- The fragments are analyzed using a special microscope that detects which fluorescent labeled base is added to the DNA template by polymerase
What is bioinformatics?
Sequencing and storing entire genomes of various model organisms in databases that can be referenced by researchers all over the world
What can bioinformatics databases be used for?
Comparing the sequences of two individuals to find out more about diversity and diseases
What is the ultimate goal of bioinformatics?
Personalized healthcare
Define:
BLAST
Basic Local Alignment Search Tool
* Method used to compare amino acid sequences
Why is comparing amino acid sequences better than comparing DNA sequences?
Genetic code is degenerate
True or False:
BLAST can find exact matches between sequences
True
True or False:
BLAST can’t find near matches, only exact matches
False, BLAST can find near matches between sequences with related amino acids
True or False:
BLAST can measure the similarity between two sequences
True
BLAST can measure the similarity between two sequences, meaning…
- Can reveal new members of gene families
- Can give clues to the evolutionary tree
BLAST can find ———- —– ——- between two proteins
Structural motif matches
In yeast and bacteria, how can BLAST be used?
Can be used to find new genes by scanning for Open Reading Frames (ORFs)
How do researchers find new human genes?
Using a combination of BLAST, cDNA sequences, and other algorithms
True or False:
Biological complexity is a measure of how many genes an organism has
False, biological complexity is NOT a good measure of how many genes an organism has
What leads to higher protein diversity in higher eukaryotes?
Alternative splicing
Why is biological complexity not a good measure of how many genes an organism has?
- Alternative splicing
- Post-translational modifications
- More cells in organism, the more ways those cells can interact
