Sequencing Genomes

Question 1

Whole genome shotgun approach steps

Answer 1

1. Extract DNA from human cells
2. Cut DNA into small, overlapping fragments (contigs) using restriction enzymes (reaction is performed at suboptimal conditions- enzymes don’t cut at every site)
3. Clone contigs to make genomic library
4. Sequence each clone using Sanger sequencing
5. Use computers to reassemble sequences of contigs by puzzling together overlapping sequences
6. Deposit sequences into database, NCBI GenBank

Question 2

Other names for Sanger sequencing

Answer 2

Dideoxy sequencing or chain terminating sequencing

Question 3

Designing a primer for an unknown sequence

Answer 3

Create universal primer that anneals to MCS of cloning vector just upstream of unknown sequence

Question 4

Dideoxynucleotide (ddNTP)

Answer 4

Nucleotide that is lacking -OH group on 3’ carbon of pentose sugar
When polymerase adds this, sequencing reaction ends (can’t add nucleotide to it)

Question 5

Sanger sequencing steps

Answer 5

1. Cloning vector containing DNA of interest is amplified by PCR
2. When polymerase randomly adds ddNTP with attached fluorescent probe, sequencing reaction stops
3. Different sized segments of DNA are created
4. Segments are separated by size using capillary gel electrophoresis (electrophoresis in a capillary tube)
5. Detector identifies ddNTPs based on their unique fluorescence
6. Sequence is recreated by reading ascending order of ddNTPs

Question 6

of base pairs in human genome

Answer 6

3.2 billion

Question 7

of protein-coding genes in human genome

Answer 7

20,000

Question 8

% of genome that is genes

Answer 8

2%

Question 9

% of sequence that is common amongst people

Answer 9

99.9%

Question 10

Pseudogene

Answer 10

Gene that has lost protein coding ability through mutation

Hard to tell this apart from gene and dubious ORF

Question 11

Dubious ORF

Answer 11

Gene that hasn’t necessarily been mutated, but still produces no product
Hard to tell this apart from gene and pseudogene

Question 12

Annotation

Answer 12

Identification and description of genes and their important sequences
Can be done using computer algorithms to predict open reading frames: look for promoters, start sites, and polyadenylation sites

Question 13

Next generation sequencing/pyrosequencing steps

Answer 13

1. Extract DNA
2. Cut into contigs
3. Affix DNA to solid support (bead, chip, etc.)
4. One by one, wash dNTPs across the DNA
5. If known dNTP is incorporated by polymerase, then light is emitted
6. Reassemble overlapping sequences

Question 14

DNA marker

Answer 14

Specific region of DNA that varies among individuals

Used to create a detailed map of the individual’s genome

Question 15

Haplotype

Answer 15

Set of SNPs that are close together on a chromosome

SNPs are inherited together: low recombination rates

Question 16

Haplogroup

Answer 16

Group of individuals that share a common ancestor

Individuals have similar haplotypes

Question 17

Tag SNP

Answer 17

SNP used to represent an entire haplotype

Question 18

SNP chips or arrays

Answer 18

Method that allows for analysis of thousands of SNPs at once

Question 19

Haplotype map (hapmap)

Answer 19

Collection of all combinations of haplotypes present in a population
Used to study inheritance of complex traits and evolutionary relatedness