DNA sequencing & marker analysis

Question 1

Which type of nucleotide does Sanger sequencing use?

Answer 1

Terminated nucleotide (ddNTP) which has an H instead of an OH

Question 2

What are the advantages and disadvantages of sanger sequencing?

Answer 2

Advantages:
-robust
-relatively long sequence reads (> 1Kb)

Disadvantages:
-hard to parallelize
-cannot do very long reads

Question 3

Illumina sequencing

Answer 3

determine order of nucleotides in small targeted genomic regions or entire genomes; reversible terminated nucleotide; incorporate all four nucleotides, each label w/ a different dye; wash, four color imaging; cleave dye and terminating groups, wash

Question 4

What are the advantages and disadvantages of illumina sequencing?

Answer 4

Advantages:
-high parallelization
-all reads have the same length
-relatively accurate for short reads

Disadvantages:
-short reads

Question 5

SMRT sequencing

Answer 5

zero mode waveguide creates evanescent field; observing very small reactions; slowed down nucleotides

Question 6

What are the advantages and disadvantages SMRT sequencing?

Answer 6

Advantages:
-long reads
-error profile is truly random

Disadvantages:
-high error rate
-expensive and big
-hard to parallelize

Question 7

Summary of all three sequencing.

Answer 7

Sanger sequencing:
-terminated nucleotides
-measure length

Illumina sequencing:
-reversible terminated nucleotides
-take photo every cycle and observe color

SMRT sequencing:
-slowed down nucleotides
-take movie in real time

Question 8

Nanopore sequencing

Answer 8

allows sequencing to be down anywhere

Question 9

DNA polymorphisms

Answer 9

two major classes: single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs)

Question 10

DNA markers

Answer 10

used to identify linked markers to traits; used for genetic and physical mapping of chromosomes

Question 11

Single nucleotide polymorphisms (SNPs)

Answer 11

base pair differences between individuals; account for 90-95% of sequence variation and arise by spontaneous mutation; most common type of DNA polymorphism; may or may not change a codon

Question 12

Short tandem repeats (STRs)/micro-satellite sequences

Answer 12

contain very short (2-6bp) tandem repeats; repeated a few times and up to 100 times; are highly polymorphic; distributed widely in the human genome w/ thousands of sites currently known; STRs are usually typed by PCR w/ primers flanking w/ sequence; population may have different allele lengths for STRs; an individual may be either homozygous or heterozygous for a particular STR

Question 13

What does each human show?

Answer 13

A unique combination of STR polymorphisms

Question 14

DNA typing

Answer 14

population studies to determine variability in groups of people; proving horse pedigrees for registration purposes; forensic analysis in wildlife crimes, allowing body parts of poached animals to be matched and used as evidence; detection of pathogenic nematodes in foods such as fish;