Genomic Sequencing

Question 1

Why Sequence genomes?

Answer 1

• To understand genetic variation with respect to phenotypic variation
• Inheritance
• Comparative genomics (ancestry/evolution)
• Forensics
• Understand genetics of extinct species
• Gives insight into normal functions of genes
• Pharmacogenomics: Tailored drug treatments for specific genome

Question 2

To sequence the human genome:

Answer 2

Whole Genome Shotgun Approach

-Mass cloning of fragments into cloning vectors.

Question 3

Whole Genome Shotgun Approach Step 1

Answer 3

Extract DNA from cells

Question 4

Whole Genome Shotgun Approach Step 2

Answer 4

Cut DNA into small, overlapping fragments with restriction enzymes

• Rxn performed in suboptimal conditions, which don’t let enzymes to cut all sites
• This is why fragments overlap
• Fragments are called “contigs” for continuous sequence

Question 5

Whole Genome Shotgun Approach Step 3

Answer 5

Clone contigs into a cloning vector to make a genomic library.

Question 6

Whole Genome Shotgun Approach Step 4

Answer 6

Sequence each clone using Sanger Sequencing technique

Question 7

Whole Genome Shotgun Approach Step 5

Answer 7

Use computers to reassemble sequences of the contigs by puzzling together the overlapping sequences

Question 8

Whole Genome Shotgun Approach Step 6

Answer 8

Deposit sequence information into NCBI GenBank Database

-Public can use this because it’s paid for by tax dollars.

Question 9

• AKA “Dideoxysequencing or Chain Terminating Sequencing”
• Based on DNA replication/PCR of a DNA template (what you want to sequence)
  
  • Can be circular or linear
• Polymerase adds nucleotides starting from a primer based on complementary sequences

Answer 9

Sanger method of sequencing

Question 10

If you don’t know the sequence, how can you design a primer?

Answer 10

Use a universal primer.

1) Can’t design a primer against an unknown sequence
2) Can have a universal primer that can be used for all clones.

Question 11

Deoxynucleotide vs. Dideoxynucleotide

Answer 11

• Deoxynuc. has OH group on 3’ C, can have phosphodiester bond
• Dideoxynuc. has H on 3’ C, cannot make phosphodiester bond
• Incorporation of ddNTP causes synthesis of that new strand to stop

Question 12

What’s happening in the PCR tube?

Answer 12

There are:

• polymerase, plasmid, primer, dNTPs, ddNTPs
• fluorescent molecules tag end of sequences

Question 13

After reaction is complete

Answer 13

Array of products with fluorescent molecules attached are separated by size, using a process called capillary gel electrophoresis

Question 14

Gel-filled capillary

• when charge is applied, larger products congregate at top and smaller products congregate at bottom
• Smaller products come off from bottom which is when fluorescent molecules are detected.

Answer 14

Capillary Gel Electrophoresis

Question 15

Reading a capillary gel electrophoresis

Answer 15

• different colored peaks represent a different base
• read the sequence by the order of the colored peaks
• can be some overlap
• read left to right

Question 16

Final Step: reassembling the sequence

Answer 16

Repeat Sanger sequencing for each clone in the library and then reassemble the contigs using overlapping sequnces.

Question 17

Things we have learned:
-The sequences of “simpler” organisms like yeast, bacteria, flies, and mice
-3.2 billion basepairs
-About 20,000 protein coding genes
-About 5,000 genes do not code for protein
code for: microRNA, exRNA, tRNA, rRNA, etc…

Answer 17

• Introns are large (can be >100kb)
• Genome is only 2% genes (but 98% isn’t junk!)
• Average gene is 3,000bp (largest is dystrophin=2.4million bp)
• Genes are clustered together on chromosomes
• People have 99.9% of their sequence in common.

Question 18

What we haven’t learned:

• Long stretches of repeated DNA sequences that were hard to reassemble
• genes vs. pseudogenes vs. dubious ORFs

Answer 18

-What a gene product actually does
Can find out by:
-compare to a known gene product
-mutate gene product and study it

Question 19

Looks like a gene but doesn’t make a gene product.

Answer 19

Dubious ORFs

Question 20

Mutated so much that it can no longer make anything.

Answer 20

Pseudogene

Question 21

How do we find protein coding genes (versus all the other sequences in the genome?)

Answer 21

1. Compare the cDNA library to genomic library

2. Use computer algorithms to look for consensus sequences.

Question 22

Use computer algorithms to predict Open Reading Frames (ORFs)
-Looks for TATA, Start, Stop, certain percentage of GC (genes tend to have more GC than noncoding regions)

Answer 22

Use of Computers to annotate genes

Question 23

Identification and description of genes and their important sequences
Goal: assign functions to all of the genes of an organism
-Understand variation w/in and among organisms
-Identify where traits come from

Answer 23

Annotation

Question 24

Alternative sequencing to Shotgun sequencing

Answer 24

1. Next generation sequencing
2. Exome sequencing
3. Analyze genetic markers throughout the genome (SNPs)

Question 25

• Fast and Cheap sequencing method

- Pyrosequencing

Answer 25

Next Generation Sequencing

Question 26

General steps for Next Gen. Sequencing

Answer 26

1. Extract DNA
2. Cut to overlapping contigs
3. affix DNA to solid support
4. one-by-one washings of dNTPs across the DNA
5. If that known dNTP is incorporated, then light is emitted
6. Reassemble by overlapping sequences

Question 27

A specific region of DNA that varies among individuals

ex. SNPs are present 1 in every 1000 bp of DNA
- used to create a detailed map of the individual’s genome.

Answer 27

DNA Markers

Question 28

Set of SNPs that are close together on a chromosome

Answer 28

Haplotype

Question 29

Within a family, haplotyes are rarely scrambled by:

Answer 29

genetic recombination

Question 30

Group of individuals that share a common ancestor because they all have similar haplotypes

Answer 30

Haplogroup

Question 31

SNP used to represent an enire haplotype

aka. diagnostic SNP

Answer 31

Tag SNP

Question 32

Is a way to look for a whole bunch of SNPs at once in a genome.

Answer 32

SNP Chips/ Array

Question 33

• More to do with a population than with individuals
• Is a collection of all the combinations of haplotypes present in a population
• Used to study inheritance of complex traits
• Used to study evolutionary relatedness

Answer 33

Haplotype map (hapmap)

Question 34

Ethical concerns?

Answer 34

• Misconceptions about genetics by the layperson?
  
  • oversight of personal genotyping services
• Insurance regulations?
• Patenting of genes?
• Are some people “better-suited” for certain careers based upon their DNA?
• Should certain people be discouraged from having children?