Chapter 2: 2.1 Sequencing

Question 1

What are the key steps to undergo if you want to sequence a genome?

Answer 1

• Purification
• Fragmentation
• Amplification
• Sequence fragments
• Re-assembly of fragments

Question 2

Define:

Purification

Answer 2

Isolating DNA from an organism or cells

Question 3

Define:

Fragmentation

Answer 3

Breaking up the genome into smaller fragments to be sequenced

Question 4

Define:

Amplification

Answer 4

Make more copies of our fragments

Question 5

Define:

Sequence fragments

Answer 5

Assigning nucleotide bases to our fragments

Question 6

Define:

Re-assembly of fragments

Answer 6

Put all the sequence fragments back together to create a continuous sequence

Question 7

List:

Types of Sequencing

Answer 7

1. Sanger Sequencing
2. Whole Genome Shotgun Sequencing
3. Next Generation Sequencing

Question 8

What is the Sanger Sequencing Method also known as?

Answer 8

The dideoxy method

Question 9

Describe:

Dideoxynucleotides

Answer 9

Have an H on the 3’ carbon of the sugar-phosphate backbone instead of OH attached to a fluorescent tag

Question 10

How does Sanger Sequencing work? (5)

Answer 10

1. Amplified fragments are replicated again in the presence of the ddNTPs
2. Replication enzyme uses normal nucleotide bases and then randomly inserts a ddNTP that stops replication
3. Fluorescently labeled sequencing fragments are run through electrophoresis to separate them by size
4. The fluorescent tags are all different colors for their respective nucleotide base, need to subject gel to fluorescent filter

Question 11

What is the order of sequence in Sanger Sequencing?

Answer 11

Shortest to longest (fragment that travelled most to fragment that travelled least)

Question 12

What is Sanger Sequencing most useful in?

Answer 12

Most useful in sequencing single genes

Question 13

What is Whole Genome Shotgun Sequencing most useful in sequencing?

Answer 13

Useful in sequencing the entire genome

Question 14

How does Whole Genome Shotgun Sequencing?

Answer 14

1. Isolate genome DNA and break up into overlapping fragments
2. Clone each fragment into a plasmid vector
3. Sequence the genomic DNA fragment in each clone
4. Use computer programs to re-align sequences based on areas of overlapping

Question 15

In Whole Genome Shotgun Sequencing:

How is genome DNA isolated and broken up into overlapping fragments?

Answer 15

By using 2 DNA samples that have been digested by 2 different restriction enzymes

Question 16

What does Next Generation Sequencing allow for?

Answer 16

Allows one sequencing instrument to carry out billions of sequencing reactions at the same time

Question 17

What is Next Generation Sequencing useful in sequencing?

Answer 17

Useful for multiple gene sequencing of whole genome sequencing

Question 18

How does Next Generation Sequencing work?

Answer 18

1. DNA fragments prepared by adding double stranded linkers to both ends of the fragment
2. Fragments are amplified using PCR primers that are complementary to linkers
3. Primers are then used to covalently bond the DNA fragments to a solid surface in a tight cluster
4. The fragments are analyzed using a special microscope that detects which fluorescent labeled base is added to the DNA template by polymerase

Question 19

What is bioinformatics?

Answer 19

Sequencing and storing entire genomes of various model organisms in databases that can be referenced by researchers all over the world

Question 20

What can bioinformatics databases be used for?

Answer 20

Comparing the sequences of two individuals to find out more about diversity and diseases

Question 21

What is the ultimate goal of bioinformatics?

Answer 21

Personalized healthcare

Question 22

Define:

BLAST

Answer 22

Basic Local Alignment Search Tool
* Method used to compare amino acid sequences

Question 23

Why is comparing amino acid sequences better than comparing DNA sequences?

Answer 23

Genetic code is degenerate

Question 24

True or False:

BLAST can find exact matches between sequences

Answer 24

True

Question 25

True or False:

BLAST can’t find near matches, only exact matches

Answer 25

False, BLAST can find near matches between sequences with related amino acids

Question 26

True or False:

BLAST can measure the similarity between two sequences

Answer 26

True

Question 27

BLAST can measure the similarity between two sequences, meaning…

Answer 27

1. Can reveal new members of gene families
2. Can give clues to the evolutionary tree

Question 28

BLAST can find ———- —– ——- between two proteins

Answer 28

Structural motif matches

Question 29

In yeast and bacteria, how can BLAST be used?

Answer 29

Can be used to find new genes by scanning for Open Reading Frames (ORFs)

Question 30

How do researchers find new human genes?

Answer 30

Using a combination of BLAST, cDNA sequences, and other algorithms

Question 31

True or False:

Biological complexity is a measure of how many genes an organism has

Answer 31

False, biological complexity is NOT a good measure of how many genes an organism has

Question 32

What leads to higher protein diversity in higher eukaryotes?

Answer 32

Alternative splicing

Question 33

Why is biological complexity not a good measure of how many genes an organism has?

Answer 33

1. Alternative splicing
2. Post-translational modifications
3. More cells in organism, the more ways those cells can interact