The Genomics Laboratory

lessfrustratedBiotechnology

Oct 23, 2013 (3 years and 11 months ago)

90 views

The Genomics Laboratory


Key Technologies


PCR


Gel electrophoresis


SNP genotyping through Microarrays


Inputs


Biological samples


Subject and pedigree information


Outputs


Genotypes (raw data)


Report phenotypes (presence of genetic defect)


Report parentage (or predictions of parentage)


Report predictions of other traits


Polymerase Chain Reaction


PCR
-

What is it?


PCR is a laboratory technique for copying a portion of a
DNA molecule


PCR can be thought of as DNA replication in
-
vitro (in a
tube) rather than in
-
vivo (in a cell / living system)


Characteristics


Powerful / Sensitive


Possible to use even in cases where there is a very
small amount of DNA available


Theoretically, with even one molecule of DNA, enough
copies can be made for scientific analysis


Enabled the biotechnology revolution


Why do we want to copy or
"amplify" DNA?


Deciphering DNA


Instrumentation capable of "reading" a DNA sequence
need a sufficient concentration of DNA to operate


This includes determining the nucleotide present at
specific locations in the genome


Can be combined with other techniques such as
tagging sequences with labels


Some applications: testing for genetic defects,
parentage, viruses, tissue typing, forensics


Why do we want to copy or
"amplify" DNA? (2)


Building sequences


Sequences can be designed and constructed
--

PCR provides
raw material


PCR provides a means of selectively copying sequences of
interest
--

such as genes


Combined with other biochemical methods, part of bioengineering
"toolkit"


Resulting sequences can be further manipulated to create
"recombinant DNA" (
rDNA
) and even inserted into a genome


Some applications that are enabled: measurement of gene
expression, production of recombinant protein products such as
insulin and HGH, genetically engineered crops


Some History...
Kary

Mullis


Invented in 1983 by
Kary

Mullis while driving his Honda Civic on the
Pacific Coast Highway 128 from San Francisco to Mendocino


"I do my best thinking while driving"


He was working out in his mind, as he drove, a process for detecting
the nucleotide at a specific DNA position


The process he was contemplating reminded me of the SNP
genotyping process that I'll be discussing later today, except that he
was working with very few DNA molecules


Known for some unconventional ideas, both before and since the
invention of PCR


The invention of PCR revolutionized molecular biology, and made
possible the sequencing of the human genome (and others)


Yet the idea is of such "utter simplicity" that the Mullis indicated the
initial response of most scientists is: "Why didn't I think of that?"


Mullis received the Nobel Prize in Chemistry in 1993, and a $10,000
bonus from
Cetus
, his employer


Cetus

later sold the PCR patent to
LaRoche

for $300M



Background: DNA characteristics
important to PCR


For replication occur, the DNA strands must separate
("denature" or "melt")


Hydrogen bonds between nucleotides much weaker
than covalent bonds on the backbone


Affinity for each nucleotide for its complement


Affinity for a sequence to bind to its complement
(and only its complement)


Replication requires a starting place: a primer


Let's review the structure of DNA and how it
replicates


DNA Components


Nitrogenous Base:



N is important for hydrogen bonding between bases



A


adenine with T


thymine (double H
-
bond)



C


cytosine with G


guanine (triple H
-
bond)



Sugar:



Ribose (5 carbon)



Base covalently bonds with 1’ carbon



Phosphate covalently bonds with 5’ carbon



Normal ribose (OH on 2’ carbon)


RNA



deoxyribose (H on 2’ carbon)


DNA



dideoxyribose (H on 2’ & 3’ carbon)


used in DNA sequencing



Phosphate:



negatively charged

credit: from bioalgorithms slide 90

Basic DNA Structure

Phosphate

Base (A,T, C or G)

http://www.bio.miami.edu/dana/104/DNA2.jpg

Sugar

DNA Replication



from bioalgorithms slide 98

DNA Replication

PCR Process


PCR similar to in
-
vivo DNA replication, with the
following differences:



Replication Step

In
-
vivo

PCR

PCR dependent
on

Melting of DNA strands

Initiated by enzymes,
especially
helicase

Increase temperature

Weak hydrogen bonds that can be broken
with moderate heat, tolerance of in
-
vitro
system to heat

Synthesis of primer

DNA primase

Oligonucleotides

synthesized in the
laboratory

Technology to
synthesize
"
oligos
"

Addition of nucleotides
("elongation")

DNA polymerase

Taq polymerase

Discovery of "taq", an enzyme found in
bacteria living only in hot
springs

PCR Reagents


DNA template


Primers (oligonucleotides)


Important: Must be designed to span fragment of
interest, on opposite strands


Taq polymerase


dNTP's


deoxyribonucleoside triphosphates


PCR buffer


w/salts


Magnesium chloride


Water


Combine into a "Master Mix"


Shaken, not stirred


PCR Process in a Nutshell

1.
Design oligonucleotide primers that span region to
be copied

2.
Add primers to reaction mixture

3.
Thermally denature the target DNA (94 deg
-
C)

4.
Reduce temperature to allow annealing of primers
and target DNA (60 deg
-
C)

5.
Increase temp. for efficient elongation (72 deg
-
C)

6.
Repeat steps 3
-
5 for 25
-
35 cycles


Taq Polymerase

Thermus aquaticus,

a thermophilic bacteria

discovered in
1969 in hot spring of Yellowstone National park

. It can
tolerate high temperature. The DNA polymerase (Taq
polymerase) was isolated.


Taq polymerase was an innovation
adde
d
subsequent to original invention of PCR process


If regular DNA polymerase is used, it is
necessary to add it at certain points as it will not
tolerate the high temperatures


Use of “taq” makes process much faster


Oligonucleotide primers


To perform PCR, 10
-
20bp sequences on both sides of the
sequence to be amplified must be known because DNA (taq)
polymerase requires a primer to synthesize a new strand of DNA


The primer must be designed to be very specific to the sequence, so
that it will only bind in one place


The term “oligonucleotide” means that the nucleotide was
synthesized


In PCR, the oligonucleotide is used as a primer


Note that the two primers are different, one for each DNA strand,
designed to bond at opposite ends of the region of interest


This design is important for producing sequences of a precise “unit”
length


Do you want to order some primers? Go to the website for
Integrated DNA technologies (for example) and specify the desired
sequence…they can be ready in a few days!!!

PCR Reaction Step 1:
Denaturation

Raise temperature to 94
o
C
to separate the duplex form
of DNA into single strands

Step 2: Annealing


Anneal primers at 50
-
65
o
C

Step 3: Extension



Extend primers: raise temp to 72
o
C, allowing Taq
pol to attach at each priming site and extend a
new DNA strand

Repeat


Repeat the Denature, Anneal, Extension steps
at their respective temperatures…

Polymerase Chain Reaction


Problem
: Modern
instrumentation cannot
easily detect single
molecules of DNA, making
amplification a prerequisite
for further analysis


Solution
: PCR doubles the
number of DNA fragments
at every iteration


1… 2… 4… 8…

Question that was troubling

(to me):


How does the PCR process control the length of the
copied DNA fragments?


By the amount of time the process is allowed to stay at
the “Extension” temperature?
-
Not really


We’ll take a closer look.

Exponential increase in copies
of target DNA (well, almost!)

Cycle Number

Target

DNA

Copies

Longer Copies

Total Copies

1

0

2

2

2

0

4

4

3

2

6

8

4

8

8

16

5

22

10

32

30


1,073,741,766

60


1,073,741,826

See
http://www.youtube.com/watch?v=_YgXcJ4n
-
kQ

How is this automated?


Through the “PCR Machine”, known as a Thermal
Cycler


Here is a picture:
(search YouTube for “PCR Song”!)

Single Nucleotide Polymorphism (SNP)
Genotyping


~99% of human genomic loci are identical for all
individuals


The remaining 1% appear to account for the vast
genetic variation we observe, including in the
susceptibility of individuals to disease


A SNP is a specific locus on the genome where there is
variation in a significant portion of the population


Variation at a single base pair location


Since each individual inherits one copy of DNA from
each parent, each SNP can have three allelic values,
commonly referred to as AA, BB, or AB

SNP Genotyping Methodology


Use PCR or other (perhaps similar) biochemical
method to amplify DNA


Use chemically labeled oligonucleotide primers called
“probes” to bind around or in proximity to the SNP


The label may also be attached at the SNP position
(discussed in next slides)


The purpose of the label is to allow detection of the
presence of the SNP through instrumentation


Example labels are based on detection by florescence or
by unique mass

Use of
dideoxynucleotide

(
ddNTP
)


Dideoxynucleotide

(
ddNTPs
) can be used to extend a
probe, to add a base at the SNP position


A
ddNTP

lacks an

OH (hydroxyl) at the 3’ position on
the
deoxyribose

sugar, preventing addition of
additional bases, thus terminating the chain


ddNTPs



Buy them labeled!

Single base extension of probe
in the Illumina “
Infinium

asay

Electrophoresis


A copolymer of mannose and galactose,
agaraose, when melted and recooled,
forms a gel with pores sizes dependent
upon the concentration of agarose



The phosphate backbone of DNA is highly
negatively charged, therefore DNA will
migrate in an electric field


The size of DNA fragments can then
be determined by comparing their
migration in the gel to known size
standards.

Gel Electrophoresis

SNP Genotyping with Gel
Electrophoresis

Gel Electrophoresis (continued)

Sequenom
iPlex


MassARRAY



Will use the Sequenom
iPlex

as an example of
Microarray technology for SNP genotyping


Many thanks go to Dr. Adam Shahid (Molecular
Biologist) of Pfizer Animal Genetics for describing this
technology and process


Many DNA microarray platforms have the assays and
chemistry “pre
-
packaged” and ready to detect specific
SNPs (or other DNA or RNA of interest)


The Sequenom is useful as a “general” SNP genotyping
machine, allowing development of assays for specific
SNPs of interest





Sequenom
iPlex



Assay Design


Develop assay to isolate and amplify DNA sequences
called “probes” that are terminated by the SNPs of
interest


The assay design will require that each SNP
-
terminated probe have a distinct mass (within the
experiment).


Mass
-
modified
ddNTPs

are used to facilitate this


Using the MALDI
-
TOF mass spectrometer (+
software), the SNP allele can be accurately identified
based on mass


Examples using “10
-
mer” sequences


Example that will not work (masses not
unque
)


Sequence ACGATCGAAC precedes SNP
-
X


Sequence ACGATCGAAT precedes SNP
-
Y


Note that the first 9 bases are identical between these two
sequences


In this example, the probes for SNP
-
X and SNP
-
Y would have
the same mass if SNP
-
X had an allele of T and SNP
-
Y had an
allele C


Mass (ACGATCGAAC T) = Mass (ACGATCGAATC)


(i.e., since they contain the same 11 bases)


This would be easily solved if the
two

binding sequences
were of different length


Trivial example, but becomes more complex when
considering many SNPs being assayed at once and taking
instrument tolerances into consideration

More on
Oligonucleotides


Sequenom’s

MassARRAY

Designer software
automatically designs PCR and extension primers
(probes) for each SNP to be investigated


Can be somewhat computationally intensive!


Required
oligonucleotides

then are ordered from
suppliers

MALDI
-
TOF Mass Spectrometry


Allows molecular mass readout of extended
oligonucleotides

(i.e., probes bound to SNPs of
interest)


Extended
oligos

are
crystalized

in special matrix on a
silica chip (via robotic liquid handling system), and
chip is loaded into the MALDI
-
TOF


Crystal is vaporized by a laser and
analyte

(extended
oligo
) is ionized and “flies” to the oppositely charged
end of the ionization chamber


Masses are individually detected based on flight time


Software translates masses into SNP allele readouts



The Sequenom Process (
iPlex
)

1.
Use PCR to amplify DNA fragments containing SNPs

2.
Use shrimp alkaline
phosphatase

(SAP) to neutralize
unincorporated
dNTPs

(
dephosphorylation
)

3.
Extension reaction


add
ddNTPs

4.
Resin step (de
-
salting)

5.
Spotting, loading, and running on MALDI
-
TOF mass
spectrometer

Example SNP Genotype Data

SNP Name



Sample ID

Allele1 Allele2

ARS
-
BFGL
-
BAC
-
10975

US4042705

A

A


ARS
-
BFGL
-
BAC
-
11025

US4042706

A

B


ARS
-
BFGL
-
BAC
-
11044

US4042707

B

B



Presentation #1


Tom Klomparens, CS 6030

Title: Introduction to the Genomics Laboratory



References:


Mullis, K. B., "The Unusual Origin of the Polymerase Chain Reaction", Scientific American, April 1990.


Mullis, K. B., Nobel Prize lecture,
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis
-
lecture.html


“The Invention of PCR”, at bitesizebio.com,
http://bitesizebio.com/articles/the
-
invention
-
of
-
pcr/


Russell, Peter J.,
iGenetics
, A Molecular Approach, chapter 8 (The Mapping and Sequencing of Genomes) and
chapter 9 (Functional and Comparative Genomics)


Hoppe, Pamela (WMU), lecture slides from BIOS 2500 (General Genetics)


Jones, Neil C., and
Pevzner
,
Pavel

A., An Introduction to Bioinformatics Algorithms, chapter 3, and Molecular
Biology slides at
http://bix.ucsd.edu/bioalgorithms/slides.php


“PCR”,
http://www.youtube.com/watch?v=_YgXcJ4n
-
kQ


“The PCR Song by Scientists for Better PCR”,
http://www.youtube.com/watch?v=7uafUVNkuzg&feature=related


LaFramboise
, Thomas, et. al., “SURVEY and SUMMARY: Single nucleotide polymorphism arrays: a decade of
biological, computational and
technlogical

advances”, Nucleic
Acides

Research, 2009,
Vol
, 37, No 13 (4181
-
4193)


Gabriel, Stacey, et. al., “SNP Genotyping Using the Sequenom
MassARRAY

iPLEX

Platform”,
http://jmgroup.pl/kawaska/download/SNP%20Genotyping%20Using%20the%20Sequenom.pdf


Chan, Michael, “Application of PCR and Microarray in Molecular Biology”,
www.slideworld.org


Shahid, Adam, Ph. D., Pfizer Animal Genetics, one hour interview on PCR, July 11, 2012


Shahid, Adam, Ph. D., Pfizer Animal Genetics, one hour interview on SNP genotyping, July 12, 2012