molecular biology – pcr, sequencing, genomics molecular biology techniques ii. polymerase chain...

MOLECULAR BIOLOGY – PCR, sequencing, Genomics

MOLECULAR BIOLOGY TECHNIQUES II.

Polymerase Chain Reacton – PCRDNA sequencing

Amplification of specific DNA fragments

MOLECULAR BIOLOGY – PCR

Cloning and/ or isolation from a genomic library

Synthetically derived DNA

Both possible but not the most convenient of methods e.g. cost and/ or labour intensive

Polymerase Chain Reaction (PCR)


A mechanism to exponentially amplify a specific DNA fragment in a test tube, using the principles of specific DNA base-pairing and DNA replication and

employing these in repeated cycles

* The oligonucleotide primer sequences must be complementary to DNA sequence flanking the fragment to be amplified and match with DNA sequence from the opposing strands of that fragment - see next slide

THERMAL CYCLING

~94oC - Denaturation step

~60oC - Primer annealing step

37oC - Extension step

• DNA containing fragment to be amplified (e.g. genomic DNA or cDNA)

• Two oligonucleotide primers (ss) specific to DNA sequence of desired fragment*

• Purified DNA polymerase (Klenow frag.)

• deoxyribonucleotide triphosphates (dNTPs)

• Buffer solution (with required Mg2+ and K+ cations)

x25-35

REPEATED THERMAL CYCLING - initiates new rounds of DNA replication that can use the products of the previous round as template, thus exponentially

amplifying the target DNA fragment

5’ 3’

5’3’

5’ 3’

5’3’5’ 3’

5’3’

DNApol

DNApol

primerprimer

DENATURATION 94°C

DENATURATION

DENATURATION


ANNEALING ~60oC

dsDNA FRAGMENT TO BE AMPLIFIED

EXTENSION - 37oC (Klenow)

DENATURATION

With each repeated THERMAL CYCLE (denaturation, annealing & extension) the amount of target dsDNA doubles

Yellowstone National Park Thermal Springs


PCR’s DNApol problem !

INITIAL DENATURATION

DENATURATION

ANNEALING

EXTENSION

TERMINAL EXTENSION

THERMAL CYCLING

e.g. x30

Primitive PCR machine (3 water baths)

94oC37oC60oC

INITIAL DENATURATION

DENATURATION

DNApol (Klenow fragment) is killed by the heat

Expensive Klenow had to be added after every thermal cycle !

Isolation of thermophillic bacteria:

Thermophillus aquaticus (50-80oC)

Has an extremly heat stable (t1/2 >40 mins at 95oC) DNA polymerase

Taq polymerase ideally suited to PCR!


Thermostable DNA polymerases and PCR

The isolation of Taq polymerase permitted the automation of PCR thermal cycling as fresh DNApol did not need to be added after every cycle !

HOWEVER: Taq polymerase lacks a proofreading activity (3‘-5‘ exonuclease) and high error rate

Stratgene inc. isolated a DNA polmerase from the hyperthermophilic archae (primitive bacteria) Pyrococcus furiosus found in the marine sediment associated with ocean thermal vents

Pfu polymerase is extremely heat stable (Pyrococcus furiosus optimum growth temperature is 100oC)

Crucially Pfu polymerase has proof-reading activity and has the lowest error rate of any known thermostable polymerase

DNA polymerase error rate (misincorporated nucleotide)

Klenow 1: 50 000Taq polymerase 1: 9 000

Pfu polymerase 1: 1 300 000 ! ! !

Pfu polymerase is IDEALLY suited for PCR applications where high fidelity amplification of DNA is required (although more expensive than Taq polymerase)


A typical PCR protocol

Template DNA, sequence specific sense and antisense oligonucleotide primers, thermo-stable DNApol (e.g. Taq or Pfu), dNTPs & PCR buffer

STEP TEMP TIME NOTES

INITIAL DENATURATION 94-96oC 2-3 mins. ensures all template DNA is single stranded (some DNApol require ‘hot-start’ for activation e.g. Pfu)

DENATURATION 94-96oC 0.5-2 mins. longer denaturation will ensure more single stranded DNA and better efficiency at cost of enzyme stability

ANNEALING ~60oC 0.5-2 mins. Higher temperature increase product specificity (less chance of mismatches forming) but lowers potential yield. 15-25oC < melting temperature Tm of annealed primer

EXTENSION ~72oC ~1 min/kb Taq processivity = 150 nucleotide per second (Pfu slower)

TERMINAL EXTENSION ~72oC 5-10 mins. Allows any incomplete products get finished

x25-30

Cetus Corporation

KARY B. MULLIS

1983 PCR discovery1985 published, patent pending1987 patented1993 Nobel prize

Journal of Molecular Biology Volume 56, Issue 2 , 14 March 1971, Pages 341-361

Studies on polynucleotidesXCVI. Repair replication of short synthetic DNA's as catalyzed by DNA polymerases K. Kleppe‡, E. Ohtsuka§, R. Kleppe‡, I. Molineux|| and H. G. Khorana||

Institute for Enzyme Research of the University of Wisconsin, Madison, Wisc. 53706, U.S.A.

Received 20 July 1970.

Dr. Kjell Kleppe H.G. Khorana

Mullins would have been ‘aware’ of the work of Kleppe and Khorana. Although their method did not amplify DNA it is generally accepted their research was a ‘primer’ for PCRs

discovery


‘Invention’ of PCR

http://www.sciencedirect.com/science?_ob=JournalURL&_cdi=6899&_auth=y&_acct=C000031739&_version=1&_urlVersion=0&_userid=622212&md5=4b4d2aae3ecbfe9afa78bbafd2ade0e5

http://www.sciencedirect.com/science?_ob=JournalURL&_cdi=6899&_auth=y&_acct=C000031739&_version=1&_urlVersion=0&_userid=622212&md5=4b4d2aae3ecbfe9afa78bbafd2ade0e5

http://www.sciencedirect.com/science?_ob=IssueURL&_tockey=%23TOC%236899%231971%23999439997%23524037%23FLA%23&_auth=y&view=c&_acct=C000031739&_version=1&_urlVersion=0&_userid=622212&md5=ac986eefa6607fb5db316748387a31ac

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WK7-4DKM9WR-36&_user=622212&_handle=V-WA-A-W-AY-MsSAYZW-UUW-U-AACDYAWWVY-AACCBEBUVY-CZVECUUE-AY-U&_fmt=summary&_coverDate=03/14/1971&_rdoc=10&_orig=browse&_srch=%23toc%236899%231971%23999439997%23524037!&_cdi=6899&view=c&_acct=C000031739&_version=1&_urlVersion=0&_userid=622212&md5=fa43ee4cb8c22320cb0bab02a0a81572





‘Polymerase chain reaction (PCR)’ amplification of DNA -

video/ tutorial

http://www.sumanasinc.com/webcontent/animations/content/pcr.html


http://www.sumanasinc.com/webcontent/animations/content/pcr.html


Experimental uses of PCRIntroduction of specific and useful DNA sequences

Sequence specific (i.e. complementary) DNA oligonucleotide primer with non-complementary yet useful 3’ sequence

Incorporation of useful DNA sequence into PCR product

PCR

Generation of restriction enzyme sites for cloning

EPITOPE TAG

Addition of extra protein coding DNA sequence for a ‘tag’ that can be used experimentally to detect or

purify a protein

Experimental uses of PCR


Introduction of specific mutations within recombinant DNA ‘directed mutagenesis’

3‘ CGCACGACACTACATCGACTACGACTTACGACGCTACAAGTTCATGAC 5‘

Protein coding DNA sequence (cDNA)

R T T L H R L R L T T L Q V H DQ

5‘ TGCTGTGATGT GCTGATGCTGAATGC 3‘T

Mutagenic primer



Degenerate PCR



Nested PCR: two rounds of consecutive PCR using a second pair of primers with annealing sites within the products produced by the first pair of primers

Some DNA fragments can sometimes be difficult to amplify by PCR - (potential secondary structures or spurious products arising from primers binding other on-target DNA). Nested PCR

will increase the yield of true target DNA

GCTGTGATGTAGCTGATGCTGAAT3’TCGATCGCACGACACTACATCGACTACGACTTAAGACGCTACAA’5

GCTGTGATGTAGCTGATGCTGAATG3’TCGATCGCACGACACTACATCGACTACGACTTACGACGCTACAA’5

SNP


G

amplification

CTGCGATGTT

SNP-specific primer


Detecting SNPs by PCR

Detection of SNPs is important for:

• diagnosing certain genetic diseases arising from ‘point mutation’ e.g. sickle cell anaemia (Hb gene E6V)

• identifying linkage traits e.g. SNPs in the Apolipoprotein E are associated with increased risk of Alzheimer’s diseas

Inverse PCR


A method to amplify a particular DNA region (e.g. containing a gene) with only partial

sequence information

N.B. relies on being able to cut DNA with ‘restriction’ enzymes that only cut at specific

DNA sequences - see lecture 8

DNA digested with restriction enzyme not cutting in known region

Generated compatible ends are ligated into a circle

DNA re-linearised by digestion with a

restriction enzyme recognising a site within the know

sequence

Unknown DNA can know be PCR amplified using primers specific to the known sequence at each

end

Unknown DNA can know be

PCR amplified using primers specific to the

known sequence

PREVIOUSLY UNKNOWN DNA SEQUENCE CAN BE

DETERMINED BY SEQUENCING FROM

KNOWN FLANKS

DNA SEQUENCE WILL REVEAL WHERE UNKNOWN FRAGMENTS WHERE ORIGINALLY LIGATED (i.e. LEFT AND RIGHT)

MICROSATELLITE SEQUENCES

Sequence repeats:

(A)n(CA)n(CAG)n(CAGT)n

5’ 3’3’ 5’

5’ 3’3’ 5’

a

b

Variable Number of Tandem Repeats (VNTR)

AFLP – amplified fragment length polymorphism

DNA fingerprinting


CCGAGTAGCTAGGAACTGATGAATGTCGATCGCACGACACTACATCGACTACGACTTAAGACGCTACAATCGATCGCACGACACTACATCGACTACGACTTACGACGCTACAATTGAGGTCGATGA...CCCCATGAGGGTGTGACCCGACATGACATGACATTGAGGCACAAATCAATGTAGA

AAAAAAAAAAAAAAAAAAAAAAAAA


5’

Experimental uses of PCRReverse Transcription PCR (RTPCR)

3’

mRNA

cDNATTTTTTTTTTTTTTTTTTTTTTTTTCTACATTGATTTGTGCCTCAATGTCATGTCATGTCGGGTCACACCCTCATGGGG. . .

TCATCGACCTCAATTGTAGCGTCGTAAGTCGTAGTCGATGTAGTGTCGTGCGATCGATTGTAGCGTCTTAAGTCGTAGTCGATGTAGTGTCG

TGCGATCGACATTCATCAGTTCCTAGCTACTCGG

TTTTTTTTTTT Reverse transcription

5’

3’ Normal PCR

Presence of DNA product reveals presence of mRNA in the original sample

However, more quantitative rather than qualitative results maybe required

Real-time PCR (Quantitative PCR or Q-PCR)

General PCR kinetics

PCR cycles

pro

du

ct

Plateau due to exhaustion of reagents


Measurements of abundance must be taken in the exponential

phase of the PCR

1. 2.

If the number of PCR cycles used were not in the exponential phase, one could mistake samples 1.

and 2. of being of equal concentration

Continuous measurement of product synthesis would be preferable i.e measurements in ‘real time’



SYBR green-based Q-PCR assay

• ds DNA intercalating dye

• fluoresces green under blue light

• only emits fluorescence when bound to double stranded DNA

denaturation

annealing

extension

Under PCR cycling conditions

SYBR green fluorescence can be measured at the end of either the annealing* or extension

steps after every PCR cycle and used to calculate the amount of DNA in the sample

* Measurements usually taken at the end of the primer annealing step


‘Real-time PCR (Q-PCR)’ using SYBR green-based assay - video/

tutorial

http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/real-time-pcr.html

click on this link

http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/real-time-pcr.html



Fluorescent hybridisation probe based methods (e.g. TaqMan probes)

DNA sequence complementary to DNA sequence of target molecule

Fluorescent reporter group Fluorescence quencher

+ other PCR reagents

At each ANNEALING step, probe and primers hybridises with target/ product DNA

Molecular proximity of quencher prevents reporter fluorescence

During EXTENSION step the annealed probe is digested by Taq DNApol (5’ - 3’ exonuclease activity)

Reporter fluorescence no longer quenched and used to quantify the DNA present


‘Real-time PCR (Q-PCR)’ using fluorescent molecular probes -

video/ tutorial

http://www.biosearchtech.com/support/videos/real-time-pcr-probe-animation-video.aspx

http://www.scanelis.com/webpages.aspx?rID=679



http://www.scanelis.com/webpages.aspx?rID=679

DNA SEQUENCING

MOLECULAR BIOLOGY – sequencing

(i.e. determining the order of the four possible deoxynucleotides in one of the DNA strands and by inference the order on the other strand)


Dideoxynucleotide trisphosphate chain terminator/ Sanger DNA sequencing

DNA backbone comprises phosphodiester bonds between the 5’ and 3’ carbon atoms of the deoxyribose moeities of

consecutive deoxynucleotides

Addition of an additional deoxynucleotide to a growing DNA strand, during DNA synthesis, requires a free 3’-OH group

However, incorporation of a chemically modified dideoxynucleotide (ddNTP), lacking a 3’-OH group, would prevent additional polymerisation and hence

TERMINATE DNA synthesis

Sanger realised such ‘chain termination’ could be exploited to reveal the sequence of a specific/ target DNA molecule, but how?

dGTP

dTTP

dATP

dCTP

ddGTP


Dideoxynucleotide trisphosphate chain terminator/ Sanger DNA sequencing

Target DNA, oligonucleotide primer & DNApol

3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCACGAG-5’

5’-CTGGGATACTGTACTAGC-3’

DNApol


5’-CTGGGATACTGTACTAGC







ACTTAACCTTTG

ACTTAACCTTTGATCG

ACTTAACCTTTGATCGATCTAG

ACTTAACCTTTGATCGATCTAGCCG

Generation of a series of differently sized fragments synthesised from the target DNA molecule that all end with radio-labelled dideoxy-G (specified by C in the target DNA)

ddGTP is radioactively labelled


dGTP

dTTP

dATP

dCTP

ddGTP

Target DNA, oligonucleotide primer &

DNApol

dGTP

dTTP

dATP

dCTP

ddATP


DNApol

dGTP

dTTP

dATP

dCTP

ddTTP


DNApol

dGTP

dTTP

dATP

dCTP

ddCTP


DNApol

G A T C

Repeat reaction using the three other radio-labelled ddNTPS

Now have a complete population of varying length DNA fragments (at one base-pair resolution), derived from target DNA, that end with one of four radio-labelled dideoxynucleotides


G A T C

polyacrylamide DNA sequencing gelautoradiography film

Read off DNA sequence from bottom to top (5’-3’ on newly synthesised

strand). Reverse complement for the other strand

-

+ AACACTACTTACTTAACTTAA

ACTTAACCTTTGATCGATCTAGCCG

ACTTAACCACTTAAC

ACTTAACCT

ACTTAACCTTTGATCACTTAACCTTTGATACTTAACCTTTGAACTTAACCTTTGACTTAACCTTTACTTAACCTT

ACTTAACCTTTGATCG

ACTTAACCTTTGATCGATCTA

ACTTAACCTTTGATCGATCTACTTAACCTTTGATCGATCACTTAACCTTTGATCGATACTTAACCTTTGATCGA

ACTTAACCTTTGATCGATCTAGCCACTTAACCTTTGATCGATCTAGCACTTAACCTTTGATCGATCTAG

‘Dideoxynucleotide trisphosphate chain

terminator/ Sanger DNA sequencing’ principle - videos/

tutorials

http://spine.rutgers.edu/cellbio/assets/flash/dideoxy.htm http://smcg.cifn.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/secuencia.swf


http://spine.rutgers.edu/cellbio/assets/flash/dideoxy.htm



http://smcg.cifn.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/secuencia.swf

http://smcg.cifn.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/secuencia.swf


Automation of the Sanger DNA sequencing method using fluorescently labelled ddNTPs

Each ddNTP varient is conjugated to a specific fluorescent group (ddGTP, ddCTP, ddATP and ddTTP) allowing the 4 reactions to be

pooled in one tube and the electrophoresed in the same lane

Process can be highly automated using ‘capillary tube electrophoresis’ coupled to automatic fluorescence detectors

(~1Kb max)

Principle of automated DNA sequencing

Automatic DNA sequence analyzers

capillary electrophoretic tubingdetector

The specific fluorescence signature of each band informs which nucleotide is at that position in the target DNA


How to sequence a human genome - video/ tutorial

http://www.wellcome.ac.uk/Education-resources/Teaching-and-education/Animations/DNA/WTDV026689.htm

Featuring a description of automated fluorescence based DNA sequencing

http://www.wellcome.ac.uk/Education-resources/Teaching-and-education/Animations/DNA/WTDV026689.htm

Why not try to deduce the sequence of larger segments of DNA . . .

MOLECULAR BIOLOGY – PCR, sequencing

Genes . . .

Chromosomal regions . . .

Whole Chromosomes . . .

Entire genomes?

1990 Human Genome Project(HGP)

Complete sequencing of the whole human genome within 15 years



Whole Genome Shotgun DNA Sequencing

Human genome (blood donors)

Mapping BACs to known sequence markers (i.e. identify from what part of the genome does the

BAC come from)?

Isolation of genomic DNA

Cloning of the genomic DNA fragments (i.e. to build a genomic DNA library;

consisting of BACs - 200Kb)



Fragmentation of BAC clones and BAC sub-clone libraries

(typically cloned into bacteriophage; ~2Kb)

Mapped BACs (i.e. in correct order on chromosome)

Sanger-based sequencing of the sub-clones (from either

end)

Sequence alignment of overlapping sequences from various subclones to reconstitute the

entire BAC DNA sequence



Repeated iterations of sub-clone sequencing (to give sequence depth i.e. confidence) and BAC reconstitution, for all the BACS covering the entire

genome.

GTCCTGCATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAGCTTGGCTCACATAGT

???

Human genome richin repetitive sequences:

!

Publication of a draft sequence in 2000 and a complete sequence in 2003

Francis Collins J. Craig Venter

President William J. Clinton

Now many hundreds of different species’ genomes have been

shotgun sequenced


The politics of sequencing the human genome !!!

Founded as an international publicly funded consortium effort to sequence all the bases of the human genome with 15 years at a cost of $3 billion

Aimed to provide free and open access to all the data as a

resource for research biologists

During the 1990’s a number of groups had placed patents on genes that they had cloned, setting a commercial precedent/ incentive to whole genome

sequencing

J. Craig Venter – founder of ‘CELERA Genomics’

$$$$$


1998 launched a commercial bid to sequence human genome and secure gene patents

Thus, the start of a race to publish the complete genome sequence between Celera and the publicly funded HGP begun. It was eventually decided that patents on genes were not legal

but both projects ended up publishing at the same time


How the genome was ‘won’ for all of humanity and not for ‘profit’ !

Storage of the human genome DNA sequence (3.3 billion base-pairs)

3300 books of 1000 pages with 1000 bp per page

1 data CD (786 Mb; 2bits per bp)

How to sequence a human genome by shotgun sequencing - video/

tutorial

http://www.genome.gov/19519278#al-3

MOLECULAR BIOLOGY – Genome sequencing

http://www.genome.gov/19519278

NEXT GENERATION DNA SEQUENCING (NextGen DNASeq)


Ultra high throughput with many millions of sequence reads per reaction allowing genomic scale experimentation analysis in single experiments!

• Illumina (Solexa) sequencing• Ion semiconductor sequencing (e.g. Ion Torrent)• Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS)• Polony sequencing• 454 pyrosequencing• SOLiD sequencing• Ion semiconductor sequencing (e.g. Ion Torrent)• DNA nanoball sequencing• Helioscope(TM) single molecule sequencing• Single Molecule SMRT(TM) sequencing• Single Molecule real time (RNAP) sequencing• Nanopore DNA sequencing• VisiGen Biotechnologies approach

Examples of NextGen DNASeq technologies


Illumina based DNA sequencing

DNA or cDNA

Specific DNA sequence adapters

Adapters ligated to ends of fragmented (~300bp) DNA sample

2-step process:

1. ligation of the same oligonucleotides to both ends

2. PCR based amplification, adding unique DNA sequence at each end (i.e. pink and blue in figure)

DNA sample preparation

Sample DNA attachment to flow

cell surface

Sample DNA adapters base-pair with complementary oligos fixed to the surface of the

flow cell (pink or blue)

The sample DNA is therefore primed for copying resulting in a copy of the sample DNA being

immobilised to the flow cell surface (the original sample DNA is washed away)

http://www.illumina.com/



Bridge amplification

The adapter sequences (pink or blue) at the free end of the immobilised copies of the sample

DNA are free to base-pair with other neighbouring oligos that are fixed to the

surface of the flow cell

Such ‘bridge’ interactions prime another round of DNA copying,

The result is two complementary copies of the original sample DNA being immobilised to the

slide in proximity to each other




Cluster formation

Repeated cycles of bridge amplification lead to the generation of copied complementary

clusters of the original sample DNA

The flow cell surface is covered in several million dense clusters - all representing one original DNA molecule in the sample

The cluster contains copies of both strands of the original DNA (i.e. it’s complementary).

Therefore prior to cluster sequencing one strand is removed by cleaving with a restriction enzyme that recognises a

sequence within either the pink or blue adapter.

Actual sequence reaction utilizing ‘reversible chain terminator fluorescent dNTPs’




Sequencing DNA clusters one base

at a time

A mix of sequencing primers (complementary to one of the adapter sequences), DNA

polymerase and differentially fluorescent labelled reversible chain terminator dNTPs

(A, C, T and G) are added to flow cell

Depending on the first nucleotide in the cluster, a specific fluorescent reversible chain

terminator dNTP is incorporated leading to a stop in DNA synthesis!

After washing unincorporated nucleotides away, a laser excites the flow cell and detects which

of the four fluorescent chain terminator dNTPs were incorporated in each cluster on

the flow cell. i.e. decodes the first sequenced base

Once an image recording what was the first nucleotide to be incorporated in each cluster has been taken, both the fluorescent dyes and the blocking group that prevents

extension of the DNA are removed (hence ‘reversible chain terminator dNTPs) and the cycle is repeated




Sequential sequencing rounds one base at a time

Possible to get up to 50 base-pairs of good sequence but there are millions of different

clusters!


The principles of ‘illumina-based’ next generation based sequencing -

video


http://www.illumina.com/technology/sequencing_technology.ilmn

http://www.illumina.com/technology/sequencing_technology.ilmn


http://www.youtube.com/watch?v=77r5p8IBwJk

The principles of ‘illumina-based’ next generation DNA sequencing -

video

http://www.youtube.com/watch?v=77r5p8IBwJk

ION PERSONAL GENOME MACHINE SEQUENCER

http://lifetech-it.hosted.jivesoftware.com/videos/1016

NextGen DNASeq Ion Torrent - video/ tutorial

http://lifetech-it.hosted.jivesoftware.com/videos/1016

Craig Venter Institute Sorcerer II expedition


„Our researchers discovered at least 1,800 new species and more than 1.2 million new genes from the Sargasso Sea“

Intensive horizontal gene transfer


molecular biology – pcr, sequencing, genomics molecular biology techniques ii. polymerase chain...

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