Genomic Sequencing Flashcards Preview

Bio 233 > Genomic Sequencing > Flashcards

Flashcards in Genomic Sequencing Deck (34)
Loading flashcards...
1
Q

Why Sequence genomes?

A
  • 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
2
Q

To sequence the human genome:

A

Whole Genome Shotgun Approach

-Mass cloning of fragments into cloning vectors.

3
Q

Whole Genome Shotgun Approach Step 1

A

Extract DNA from cells

4
Q

Whole Genome Shotgun Approach Step 2

A

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
5
Q

Whole Genome Shotgun Approach Step 3

A

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

6
Q

Whole Genome Shotgun Approach Step 4

A

Sequence each clone using Sanger Sequencing technique

7
Q

Whole Genome Shotgun Approach Step 5

A

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

8
Q

Whole Genome Shotgun Approach Step 6

A

Deposit sequence information into NCBI GenBank Database

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

9
Q
  • 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
A

Sanger method of sequencing

10
Q

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

A

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.

11
Q

Deoxynucleotide vs. Dideoxynucleotide

A
  • 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
12
Q

What’s happening in the PCR tube?

A

There are:

  • polymerase, plasmid, primer, dNTPs, ddNTPs
  • fluorescent molecules tag end of sequences
13
Q

After reaction is complete

A

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

14
Q

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.
A

Capillary Gel Electrophoresis

15
Q

Reading a capillary gel electrophoresis

A
  • 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
16
Q

Final Step: reassembling the sequence

A

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

17
Q

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…

A
  • 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.
18
Q

What we haven’t learned:

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

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

19
Q

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

A

Dubious ORFs

20
Q

Mutated so much that it can no longer make anything.

A

Pseudogene

21
Q

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

A
  1. Compare the cDNA library to genomic library

2. Use computer algorithms to look for consensus sequences.

22
Q

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)

A

Use of Computers to annotate genes

23
Q

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

A

Annotation

24
Q

Alternative sequencing to Shotgun sequencing

A
  1. Next generation sequencing
  2. Exome sequencing
  3. Analyze genetic markers throughout the genome (SNPs)
25
Q
  • Fast and Cheap sequencing method

- Pyrosequencing

A

Next Generation Sequencing

26
Q

General steps for Next Gen. Sequencing

A
  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
27
Q

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.

A

DNA Markers

28
Q

Set of SNPs that are close together on a chromosome

A

Haplotype

29
Q

Within a family, haplotyes are rarely scrambled by:

A

genetic recombination

30
Q

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

A

Haplogroup

31
Q

SNP used to represent an enire haplotype

aka. diagnostic SNP

A

Tag SNP

32
Q

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

A

SNP Chips/ Array

33
Q
  • 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
A

Haplotype map (hapmap)

34
Q

Ethical concerns?

A
  • 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?