Basic Molecular Approaches: Cloning and Beyond...1 Basic Molecular Approaches: Cloning and Beyond...

1

Basic Molecular Approaches: Cloning and Beyond

PHOP 6241/6242 Fall 2018 Deborah Otteson, Ph.D.

UH College of Optometry

2

2

The Human Genome Project: What It Means for You

Genetics is becoming an important factor in the diagnosis and treatment of all kinds of disorders.

4

Applications of Molecular Biology and Genetics in Medicine

• Understand processes regulate normal and abnormal function incells and tissues

• Identify and understand genetic factors that cause disease

• Predict susceptible individuals/prevention

• Develop gene replacement/corrective therapies

• Identify new drug/treatment targets

• Identify patients who will benefit from different therapies (differentcauses = different treatments)

3

5

Developing New Treatments: Understand the Biological Processes Involved in Disease

ARMDGenetic/environmental Death of photoreceptors Defects in RPE Excessive vascular growth

Dry Eye

Genetic/environmental

Nerve damage

Defects in tear production

Hormonal

KeratoconusGenetic/environmental Disintegration of Bowman’s membrane Excessive protease activity

Glaucoma

Genetic components

Death of ganglion cells

Increased IOP

Defects in aqueous production/outflow

Comorbidity (diabetes?)

6

What is Molecular Biology?

A powerful approach for studying

• Cellular/biological processes at the molecular level• Biochemical processes within biological systems

• Molecular Biology tends to be “gene” based

4

“Central Dogma” of Gene Expression and Protein Synthesis

2-7

RNA viruses: Reverse transcription(e.g. HIV, retroviruses)

2-8

Chromosomes

• Visible at the light microscopic level in cells

during mitosis

• First named in 1888

• stained by ‘new’ synthetic chemical dyes

• chroma, "color" and soma, "body”

• Number of chromosomes varies between

species.

Human: Diploid = 46 chromosomes

(23 from each parent)22 autosomes

X & Y (sex chromosomes)

Examples of Diploid chromosome #:

Flies: 8 Mice: 40

Frogs: 26 Duck: 80

Asparagus: 30 Armadillo: 64

Yeast: 32 Sweet Potato: 90

Fluorescently stainedHuman chromosomes

5

2-9

Chromosomes contain Genes

• The term ‘gene’ was coined in 1909 as the

“unit of genetic inheritance”

• VERY Controversial at the time

• 1933: “There is no consensus opinion

amongst geneticists as to what the

genes are—whether they are real or

purely fictitious.” T. H. Morgan

• 2017: We now know that the genes are

genetic units in the DNA that code for

the RNAs and proteins necessary for

cell function and structure

• Humans: 20-25K protein coding genes

in genome

Arrows: Fluorescently labeled genes in condensed human chromosomes

2-10

Chemical Composition of Nucleic Acids

DNA consists of 4 nitrogen-containing bases

Purines first synthesized in laboratory: ~1888

base nucleoside nucleotideadenine adenosine dATPguanine guanosine dGTP

Pyrimidines first synthesized in laboratory: ~1879

base nucleoside nucleotidecytosine cytidine dCTPthymine thymidine dTTP

Base + deoxyribose = nucleosideBase + phospho-deoxyribose = nucleotide (= nucleoside with phosphate)

6

Nucleotides = Deoxyribose sugar + Base (Purine or Pyrimidine) + Phosphate group

2-11

Deoxyribose

5

4 1

3 2

Carbon atoms of ribose molecules are numbered

Base attaches at #1 carbon

Deoxyribose lacks oxygen molecule on #2 carbon (H instead of OH)

Phosphate groups attached to #5 carbon

5’ and 3’ carbons are where nucleotides are joined during DNA synthesis (more on this later)

7

Watson and Crick deduced the structure of DNA using data from Franklin and Wilkins (1953)

2-13

X-ray crystallography data

• Helical DNA structure

• Base pairing: fit previously

• Determined ‘rules’ of DNA

composition (A=T, C=G)

X‐ray diffraction photograph of DNA fiber

at high humidity (Franklin and Gosling,

1953b). Interpretation of the helical‐X and

layer lines added in blue.

Watson‐Crick model of DNA, adopted from

(Watson and Crick, 1953b), with the helical

repeat associated with the layer lines

labeled. Image from: DNA Structure:

Alphabet Soup for the Cellular Soul

2-14

Watson, Crick and Wilkins:Nobel Prize in Physiology for Medicine 1962

Watson and Crick with model of double helix

(very short paper published in Nature, 1953)

8

2-15

Double-stranded alpha helix

One full turn of helix ~10 base pairs

DNA base pairs more exposed at major groove

Regulatory proteins preferentially bind to major groove (more on thislater)

majorgroove

minorgroove

The structure of DNA

3.4Å

34Å

Watson-Crick Pairing

Invariate pairing bases

• Each base pair consists of

• 1 purine and 1 pyrimidine

T – A2 hydrogen bonds

C – G3 hydrogen bonds

Pyrimidines Purines

Hydrogen bonds

9

DNA: Primary Structure

Anti‐parallel orientation of 2 strands

5’‐CAGTGATTACA‐3’3’‐GTCACTAATGT‐5’

Phosphodiester bonds between deoxyribose sugars on backbone

= nucleotide

2-18

Semiconservative DNA replication

Meselson and Stahl, 1958

• Each parent strand serves as thetemplate for synthesis of a new daughter strand

• Invariant base pairing keeps sequencethe same in new strand

• Newly synthesized strand is basepaired with a parent strand

• During mitosis, each daughter cellreceives chromosomes each containing one original and one new strand of DNA

10

2-19

How much DNA in a human?

Human cells contain23 pairs of chromosomes

20,000-25,000 genes

6 x 109 base pairs

~ 2 meters of DNA in one human CELL(if it could be stretched out !)

Total length of DNA in ONE human(> 10 trillion cells)

~20 trillion meters~ 20 billion km

Distance: earth to sun: ~150 million km

One person’s DNA would go from earth to sun and back ~66.7 times!!!!

These calculations don’t include DNA from:

Mitochondria Microbes in/on the body

2-20

Forms a bead-like structure (11 nanometer diameter) = chromatin

Chromatin condenses into 30 nm fibers

Eukaryotic DNA: Higher Order Structure

DNA double helix coils around the histonehetero-octomer (2 times, 146 bps) = nucleosome

Histone modifications (acetylation and methylation) regulate chromatin packing

11

2-21

Chromatin is organized onto protein scaffold

Packing density increases in mitotic chromosomes

Smallest human chromosome is 2 micrometers long when condensed.

Eukaryotic DNA Higher Order Structure

3-22

Transcription: DNA to RNA

• Transcription occurs in both the nucleus and mitochondria• Genomic organization and transcription/translation processes in

mitochondria are similar to prokaryotic cellsFigure 1-13

12

3-23

RNA vs DNA

RNA DNASingle stranded Double stranded

Anti-parallel strands

A, C, G, U A, C, G, TRibose sugar Deoxyribose sugar

5’ 3’ 5’ 3’

5’3’

Sense strand/non-template

Anti-sense strand/template

(RNA) (DNA)

3-24

13

3-25

Classes of RNA

Protein coding RNAs: Messenger RNA (mRNA)

• transcribed from genes in DNA

• used as template for protein synthesis

Functional RNAs : tRNA, rRNA, snRNA, miRNA

• not translated into protein

• RNA is active component

Genomic RNA

• Retroviral genome

3-26

Functional RNAs

Transfer RNA (tRNA)

• brings the amino acid to the ribosome during proteinsynthesis (translation)

Ribosomal RNAs (rRNAs)

• components of the ribosomes

Small nuclear RNAs (snRNAs)

• multiple functions

• guide the modification of mRNA

• combine with proteins to form the ribonucleoproteinprocessing complex (spliceosome)

14

3-27

RNA regulation of gene expression:

Micro RNA (miRNA)

• Encoded in genome (can be individual miRNA genesor within introns of protein coding genes)

• Contains complementary sequences to mRNA target

• Bind to the 5’ untranslated region of mRNA target

• Targets RNA for degradation by cell

Small interfering RNA (RNAi)

• Anti-viral mechanism in lower eukaryotes

• RNA binds to the mRNA at the translation start site

• Blocks initiation of translation/protein synthesis

• Used in research to alter gene expression

More Functional RNAs

RNA transcription and protein synthesis take place in different parts of the cell

Griffiths. Figure 8-11

• RNA transcription• The generation of RNA from a DNAtemplate• Occurs in the nucleus•Regulated process

transcription factorsRNA polymerasechromatin structure

Protein coding RNAs are processed in nucleus:

• capping• splicing• poly‐adenylation

Processed mRNAs are transported out of the nucleus

Protein synthesis (translation) occurs in cytoplasm

15

Adjacent genes can have different orientations in the chromosome

3-29

Coding strand for gene 1

Coding strand for gene 3

Template strand for gene3

3-30

Eukaryotes: Gene Organization

Promoter region:• binding sites in DNA sequence interact with:

• RNA polymerase• transcription factors• Other DNA modifying enzymes

• +1 is transcription start site (TSS)• Bases in DNA sequence are numbered relative to TSS

• promoter starts at -1 and goes to left (-1, -2, -3 etc)• transcribed region starts at +1 and goes to right

16

3-31

Eukaryotes: Gene Organization

Introns = transcribed but removed during splicingExons = transcribed; remain in mRNA after splicing

Exons include both untranslated and protein coding regions

Note: Prokaryotic genes do not have introns

DNA condensedheterochromatin

Densely packed nucleosomes

Histone modifications stabilize structure (deacetylation, methylation)

DNA methylation at CpG sequences can permanently shut down expression

Transcriptional regulatory are proteins excluded from heterochromatin

DNA open = euchromatin

17

DNA condensed =heterochromatin

DNA open = euchromatin

Histones moved aside (sliding and/or disassembly)

Histone modifications open the DNA structure (acetylation)

Allows transcriptional regulatory proteins to bind to DNA

Transcription factors can recruit histone modifying enzymes RNA POLYMERASE and other components of the transcription machinery

5-34

Histones Influence Transcription

Histones: Eukaryotes

octamer containing two copies of 4 subunits

DNA wraps histone ~1.65 times to form nucleosome

(~147 base pairs)

Histone tails exposed

Modification of histone tails regulates chromatin structure (open vs. closed)

18

• RNA polymerase is recruited to the basal promoter by various

transcription factors bound to the proximal and distal promoter

• RNA Polymerase transcribes both Introns and Exons

• Results in hnRNA = heterogeneous nuclear RNA = (pre-mRNA)

Eukaryotes: Transcription Overview

hnRNA

3-36

RNA is Synthesized 5’ to 3’ Direction

• The first ribonucleotide of new RNA is the 5’ end of the RNA

molecule

• RNA is the reverse complement of template (anti-sense)

strand

• RNA sequence is same as non-template (sense) strand

• EXCEPT: RNA has U instead of T

Direction of RNA synthesis

19

3-37

RNA is Synthesized 5’ to 3’ Direction

• Template DNA strand copied from 3’ to 5’

• RNA synthesized from 5’ to 3’

• New bases are added to the 3’ end of the growing RNA

• Note that two DNA strands rejoin into double helix

3-38

Eukaryotes: RNA Processing in nucleus

Capping protects 5’ end

Splicing removes introns

AAAAAAddition of poly A tail stablilizes RNA

and signals for nuclear export

20

RNA Processing: Alternative Splicing

Multiple protein isoforms generated from a single geneDifferential exon usageRegulation of timing and tissue specificity of gene expressionCan have different functions

3-40

Eukaryotes: RNA Processing in nucleus

AAAAA

3-40

CODING REGIONUntranslated regions = 5’ UTR 3’UTR

Mature mRNA

Export from nucleus

Translation into proteins

21

4-41

Translation: RNA to Protein

4-42

20 Amino acids: Classified by structure and chemical composition

Non-polar, aliphathic (non polar, hydrophobic)Alanine Ala Glycine Gly Polar, unchargedIsoleucine Ile Asparagine Asp Leucine Leu Cysteine CysMethionine Met Glutamine GlnValine Val Proline Pro

Serine SerNegative charge Threonine ThrAspartate Asp(Aspartic Acid) Aromatic (C- ring)Glutamate Glu Phenalanine Phe(Glutamic Acid) Tryptophan Trp

Tyrosine TyrPositive chargeArginine Arg Histidine His Lysine Lys

22

4-43

• Each amino acid called “residue” (R)

• Each AA is numbered: starting at #1 at N terminal (amino end), withsequential numbering to C-terminal end.

• Co-linearity of DNA, RNA and protein sequence

• Amino acids linked by peptide bonds

• Multiple amino acids linked together form a polypeptide

• Properties of amino acid side chains confer structure and function ofprotein

Levels of protein structure: Primary

Polypeptides can bend into

regularly repeating structures

created by H-bonds between

-COOH and -NH2 groups of

different amino acid residues

4-44

Levels of protein structure: Secondary

23

4-45

Tertiary structure = 3-dimensional architecture

• Chaperon proteins in ER can regulateprotein folding.

• Proteins that are incorrectly folded arerefolded or degraded

• Stabilized by various intermolecularbonds:

• disulfide

• electrostatic

• hydrogen

• van der Waals

• Amino acids located far apart inprimary sequence can be brought close together by folding

Myoglobin

Levels of protein structure: Tertiary

4-46

Many mature proteins are composed of multiple subunits = Multimeric

Joined by various intramolecular bonds

Subunits: separate proteins, may be encoded by the same or different genes

# subunitsDimer 2Trimer 3Tetramer 4

Homodimer: 2 identical subunitsHeterodimer: 2 different subunits

Example of a tetramerHemoglobin

= 2 each of 2 subunits

Levels of protein structure: Quaternary

24

4-47

How is mRNA Sequence Translated into Amino Acids?

Genetic Code:

•Amino acids are encoded

by base triplets called

codons

• Many amino acids are

encoded by more than one

codon (degenerate)

• Codons specifying the

same amino acids can

differ at the 3rd base, known

as wobble position

Translation: RNA to Protein

Genetic CodeAmino acids are encoded by base triplets called codons

Many amino acids are encoded by more than one codon (degenerate)

Codons specifying the same amino acids differ at the 3rd

base known as wobble position

25

4-49

Compare Overlapping vs. Non-Overlapping Code

If overlapping, change (mutation) of one base could alter 3 codons

If non-overlapping, only one codon affected

Which one is actually used? Non-overlapping

Consequences of point mutations

Silent mutations: no change in protein

Missense: consequence depends on location and similarity of new amino acid to original

Alters multiple amino acids: consequences varypremature termination; loss of function, gain of function

Can alter gene expression if in regulatory regionMay be silent if not in critical region

26

4-51

Components for Translation

mRNA messenger RNA

Amino Acids

tRNAs = transfer RNA

Ribosomes

rRNA = ribosomal RNA

ribosomal proteins

tRNA

Multiple tRNAs with different

structures and anticodons

Enzymes couple amino acid to

correct tRNA molecule

Anticodon arm base‐pairs with

mRNA

Provides sequence specificity

for translation

27

Two Subunits of a Ribosome Contain

Protein and RNA Molecules

• Two ribosomal subunits

• Both contain rRNA and proteins

• Small subunit binds mRNA cap

• Scans mRNA to find translationstart site:

• identified by Kozak sequencein mRNA and AUG(methionine) start codon

• Recruits large subunit

• Translation begins

4-54

1. Initiation complex assembles at 5’ capof mRNA• 40S subunity• Met-tRNA• Initiation factors +GTP

2. Initiation complex scans 5’ end of mRNAfor KOZAK sequence and start codon

(Marilyn Kozak, 1987)

-6 +15’- GCCA/GCCAUGG-3’

3. Positions Met tRNA at AUG

Translation in Eukaryotes: Initiation

Figure 9-15

28

Figure 9-13

Key Sites of Interaction in the Ribosome

A=Amino acyl site: binds aminoacyl tRNA

P=Peptidyl site: forms peptide bond

between peptide chain and new amino acid

E= Exit site: tRNA without amino acid is released

Path of each tRNA through ribosome:A P E

4-56

Termination similar in pro- and eukaryotes

Ribosome reaches a stop codon (at A site)

Release factor (RF1) binds

There is no tRNA for STOP codon: only release factor

Disassembly of thetRNA-ribosome-mRNA complex

Peptidyl cleavage to release of nascent polypeptide from tRNA

Translation: Termination

29

4-57

Post-Translational Processing of Proteins

Protein processing begins in trans-golgi network

FoldingChaperone proteins regulate folding (see tertiary structure)

Proteolytic CleavageProteins synthesized as inactive precursorCleaved to generate active forme.g. pre-proinsulin > proinsulin > insulin

Glycosylation

Covalent attachment of carbohydrates (sugars)O-linked attached to AA with hydroxyl groupsN-linked attached to AA with amine groupsOften associated with recognition/adhesive function

4-58

Post-Translational Processing of Proteins

Methylation addition of a methyl group (CH3) e.g. calmodulin, Cytochrome C

Acetylation C-terminal acetyl group, allows addition of myristol group for membrane anchoring;

e.g. catalytic subunit of cyclic AMP-dependent protein kinase (PKA) is myristoylated

Phosphorylation often associated with intracellular signal transduction

e.g. Ligand binding to a receptor causes phosphorylation (auto-phosphorylation of receptor and/or phosphorylation of G-protein) and initiates intracellular signaling)

30

Protein Targeting: Signal sequences target proteins for secretion

4-59

Signal sequence: series of hydrophobic amino acids at N terminus of polypeptide

Targets assembled ribosome to the rough endoplasmic reticulum

Signal sequence is cleaved for secreted proteins

Most transmembrane proteins targeted to rough ER by hydrophobic transmembrane domain—not cleaved

4-60

Protein Targeting/Subcellular Localization

Nuclear Localization Signal directs protein to the nucleus. e.g. transcription factors, histones

Signal Sequence sends ribosome with new/growing polypeptide to endoplasmic reticulum. Protein is synthesized into the endoplasmic reticulum.

secreted proteins e.g. insulin, collagen.

Transmembrane proteins have signal sequences but not cleaved

other proteins destined for vesicles have signal sequences

Ubiquitination attaches Ubiquitin peptide; identified proteins for destruction; sends proteins to proteosome/endosomes

31

4-61

Protein Targeting: Ubiquitinization Targets a Protein for Degradation

32

Recomb DNA 63

Isolation of specific DNA or RNA/cDNA

• Diagnosis

• Nucleotide sequencing/gene ‘chip’ analysis; GWAS

• Identify and understand effects of mutations

•Probes for analysis of mutation or gene expression

• Transfer genes into cells/organisms for functional analysis or gene

therapy

• Generate recombinant proteins for research or therapeutics

(e.g. recombinant insulin, recombinant growth hormone)

Uses of Molecular Cloning

2-64

Transcriptome:The collection of all RNA transcripts in a cell.

The genome is identical in all

cells

Proteome: Collection of all proteins in a cell

or tissueGriffiths 10th editionFigs. 1-2 & 1-5

33

Identify the Gene Mutated in Patients with Eye Disease

Example: Age Related Macular Degeneration

Genetic studies can localize chromosomal regions associated with phenotypeNow need to identify candidate genes

‐genes that are expressed in affected tissue (retina, choroid and vasculature)

‐genes that are involved in functions affected tissue (eye development, cellular differentiation, phototransduction, angiogenesis, inflammation ???)

Online Genome DatabasesNational Library of Medicine/NIH

http://www.ncbi.nlm.nih.gov/genome/guide/human/

University of California Santa Cruz http://genome.ucsc.edu/

Ensembl http://www.ensembl.org

• GWAS or other genetic methodslocalize region associated withdisease

• Identify genes that would be likelycandidate for causing disease

• Sequence the DNA to look forpossible mutations

• Use Bioinformatics analysis of humangenome databases

• Analyze mRNA and proteinexpression

• Determine protein function

• Determine effect of mutations: whydoes it result in disease?

34

Isolation of Genomic DNA for Cloning and Analysis

Needed for genetic analysis, identification of mutation and variations

Sources of DNA

• Isolate DNA frompatient/subject– Blood

– Cheek swab

– Biopsy (e.g. cancer)

• DNA in all cells shouldbe the same **

** But see article in New York Times 9/24/13: Mosaicism in human genomeshttp://www.nytimes.com/2013/09/17/science/dna-double-take.html?pagewanted=all&_r=0

http://www.alleight.com/Products/Epicentre/BuccalAmp.htm

Agarose Gel Electrophoresis of DNA/RNA

http://www.stanford.edu/group/hopes/diagnsis/gentest/f_s02gelelect.gif

pole

35

Recomb DNA 69

Cloning and Analysis of RNA

Needed for:• identification of genes expressed in a

particular tissue/cell type.

• effects of mutation on size and stability ofmRNA

• analysis of consequences of mutation onproteins/cellular function

• cloning genes for gene replacementtherapy

Ethidium Bromide stainedAgarose gel of total RNA

28S and 18S RibosomalRNA

5S Ribosomalt-RNAmicroRNAs

mRNA

RNA Sources:• Tissue of interest

• Synthetic RNA (by machine)

• In vitro transcribed (from cloned DNA)

Recomb DNA 70

Genes and Gene Products for Analysis

Capping, Splicing Polyadenylation

CODING REGION

Protein

mRNA

cDNA: complementary DNA(copy of mRNA)

Genomic DNA

AAAA

AAAA

36

Recomb DNA 71

Analysis of RNA:Reverse Transcription Generates cDNA Copy

• Isolate total RNA from cells

• Oligo-dT primers bind to poly-A tailof mRNA

• Reverse transcriptase enzymemakes DNA copy of RNA

• RNAse removes RNA strand

• DNA polymerase synthesizessecond strand

• Results in double-stranded cDNA(complementary DNA); includescopies of all mRNAs present inoriginal sample

Analysis of Gene Expression: RT-PCR

RT = reverse transcription

PCR = polymerase chain reaction

RNA cDNA PCR with gene specific primers gel electrophoresis

Standard RT‐PCR

end point assay: gene is present/absent

qualitative, not quantitative

Quantitative RT‐PCR : “real time”

determine copy number or relative abundance of RNA (cDNA)

(e.g. compare normal vs. diseased; retina vs. brain)

uses fluorescent stains/labels to quantify PCR product synthesis

reads fluorescence level at every cycle

37

PCR: Polymerase Chain Reaction

Taq DNA polymeraseIsolated from Thermus aquaticusHeat stableSome have proofreading capability

Recomb DNA 74

PCR: Polymerase Chain Reaction

Key components: template DNA; primers; deoyxribonucleotides; Taq polymerase

38

RT-PCR Analysis of Gene Expression

Agarose gel of RT‐PCR products

cDNA from multiple tissues

RT‐PCR using primers for 6 different genes

Separated on Agarose gel

Stained with ethidium bromide

All are in retina, only F, I, J are retina‐specific (lane #6)

retina retina

Quantitative “real time” PCR

Recomb DNA 76

• PCR with addition of DNA intercalator dye (SYBR green)

• Dye fluoresces only when bound to double stranded DNA

• Detector measures increase in fluorescence after each cycle

• Each cycle doubles the amount of fluorescent PCR product

From: http://eng.bioneer.com/products/GeneExpression/qPCRArrayService-detection.aspxhttp://www.thermoscientificbio.com/qpcr-master-mixes-and-assays/dynamo-hs-sybr-green-qpcr-kit/

39

Recomb DNA 77

Can also start with RNA and generate cDNA for template

Analyze by PCR or amplify targets for cloning

PCR products are often cloned into plasmids

Basic Molecular Methods: Restriction Enzymes

Bacterial enzymes

Normal function : defend against phage infection

Recognizes and cuts (digests) specific DNA sequences

e.g., EcoRI (from E. coli)

5’GAATTC 3’ 5’G AATTC 3’ 3’CTTAAG 5’ 3’CTTAA G 5’

Recognition sequences methylated in the bacterial genome to

prevent DNA digestion by restriction enzymes made by the cell

Wide range of enzymes with different target recognition sites

Commercially available

40

Cloning Using Restriction Enzymes

Cut plasmid and insert DNA with same restriction enzyme

Generate compatible “sticky ends”

Hybridize DNA fragments

Bacterial DNA ligase repairs nicked ends

Grow plasmid in E. coli

Isolation of Plasmid Clones

41

Vector DNA

= Insert (cloned) DNA

Looking for DNA Insert in Plasmid Clones

•Restriction Enzyme digest

•Gel electrophoresis

•Fluorescent stainEthidium bromide = DNA intercalator

42

Recomb DNA 83

DNA SequencingChain Termination Method (Sanger Method)

Components:

• DNA template (genomic DNA, cDNA)

• ONE primer for target

• dNTPs

• ddNTPs (each with radioactive or fluorescent label)

• Taq DNA polymerase

• Amplify by PCR– linear amplification with single primer!

• Each cycle, after a ddNTP is incorporated, DNA synthesis stops

• Generates a collection of all possible DNA fragments with ddNTP at3’ end

• Run gel to size fractionate

• Read sequence by size of each PCR product: speed and distance ofmigration determined by size

Recomb DNA 84

Di-deoxy sequencing method

Di-deoxyNTP (ddATP; ddCTP ddGTP ddTTP)

• Lacks 3’-OH group

• Cannot form a phosphodiester bond with

the next nucleotide in the growing chain

• DNA synthesis is terminated

Video of sequencing:https://www.youtube.com/watch?v=jFCD8Q6qSTM


43

• Each ddNTP (ddATP, ddTTP, ddCTP, ddGTP) is labeled with differentfluorescent molecule

• Generates all possible fragments with each labeled at one end• Shown: DNA Fragments generated by di-deoxy sequencing using ddATP

with fluorescent tag ( )

Primer


Recomb DNA 86

Automated Chain Termination Sequencing

• Automated sequencer uses electrophoresis that is performed in agel that is inside a thin capillary tube

• Reads fluorescent signal as each product passes detector

• Electropherogram (below) shows fluorescence intensity for eachPCR product

• Each peak shows a fragment with a specific ddNTP at the end

• Read sequence from left to right (small to large fragments)

44

“Next Generation” Sequencing (massively parallel sequencing)for genome and transcriptome

analysis

Recomb DNA 87

Goldman &. Domschke (2014) Making sense of deep sequencingDOI: http://dx.doi.org/10.1017/S1461145714000789 1717-1725

Analyzes all DNA or cDNA in the sample simultaneously

Each fragment 100-500 bp

Total DNA sample is tagged with an adaptor at each end

DNA is attached to “flow cell” or other substrate

The adapter hybridizes to primer for limited PCR to amplify each molecule to generate ‘spot’

Recomb DNA 88

• Uses 4 terminator NTPs that aremodified to stop elongation (likeddNTP)

• Allows addition of one baseadded at a time

• Treat entire flow cell with mix ofall 4 terminators

• Record fluorescence of everyspot on entire flow cell(photograph) after each base isadded

• Chemical treatment removesfluorescent tag and reversesterminator

• Add another batch of the 4terminated

• Repeat

Informative Video:http://www.yourgenome.org/video/sequencing-at-speed

45

Mapping RNA seq data to genome

Recomb DNA 89

• Number of aligned fragments shows relative abundance of differenttranscripts

• Can identify alternative splicing (variable abundance of differentexons)

Recomb DNA 90

Analysis of DNA, mRNA and Protein

Gene (DNA)—Southern blot

Transcription (RNA)-northern blot

Translation (protein)-western blot

General strategy:• Isolate appropriate cellular

component

• Size fractionate by gelelectrophoresis

• (electrical current drivesmolecules through gel)

• Detect using labeled probes(piece of gene of interest;antibody against protein)

• Analyze: compare wild-type vs.experimental, disease vs. healthy,etc

46

Applications of Southern blots

• Identification of a single gene in a pool of DNA fragments

• Detection of specific DNA sequences in a genome

• Studying genomic deletions, duplications, and mutations that cause various diseases

• Genotyping (e.g. transgenic mice)

• Detection of genetic diseases and cancers, such as monoclonal leukemia and sickle cell mutations

• DNA fingerprinting and forensic tests such as paternity testing and sex determination

Recomb DNA 91

Applications of northern blots

• Determine actual size of transcript(s) and reveal presence of alternative splicing in a single gene

• Gene expression studies – e.g. to observe overexpression of cancer-causing genes and gene expression in case of transplant rejection

• In diagnosis of several diseases, e.g. Crohn’s disease

• For detection of viral microRNAs that play key roles in viral infection

• To screen recombinants - by detecting the mRNA formed by the transgene

• Requires that you have RNA from the tissue where it is expressed!!!!

reverse-transcriptase PCR (RT-PCR) can provide a faster and more sensitive alternative (next slide)

Recomb DNA 92

47

Recomb DNA 93

• Start with cDNA from multiple tissues (this was done in mouse!)• RT-PCR using primers for 6 different genes• Separated on agarose gel and stained with ethidium bromide• All are present in retina, but only F, I, J are retina-specific (lane #6)

#6 = retina #6 = retina

Analysis of Gene Expression: RT-PCR of cDNA

Assess differences or changes in RNA expression between:

• Different tissues (e.g.retina vs. brain)

• Normal vs. disease state

• Response totherapy/drug treatments

Microarrays

Recomb DNA 94

Commercially available

• Tens of thousands of spots,each containing many copiesof single stranded DNAprobes

• For gene expression:contains DNA copies (cDNA)for known or predicted genes

• For GWAS/genotyping: allpossible sequence variantsof SNPs

• For mutation analysis: allpossible sequence variantsfor mutations in diseaseassociated genes

http://www.stemcore.ca/services/microarrayshttp://www.ur.umich.edu/0506/Oct17_05/02.shtml

Expected sequence: A T G C A G C C T C T G T C A GVariant (SNP, mutation) A T G C A G C C T T T G T C A G

https://en.wikipedia.org/wiki/DNA_microarray

48

Analysis of Gene Expression: in situ Hybridization

Purpose: Determine the cellular expression of RNA within a tissue

Tissue: frozen or paraffin sections

Probe: anti‐sense RNA (complementary to endogenous RNA)

make by in vitro transcription

labeled with digoxygenin protein‐tagged ribonuclotides

Hybridize probe to RNA in tissue

Antibody detection of protein‐tag; colorimetric detection of antibodies

In situ hybridization to mouse retina

Rhodopsin CRX negative control

Applications of western blots

• Medical diagnosis (some examples listed)

– HIV infection,

– Hepatitis B Virus infections

– Bovine Spongiform Encephalopathy (“mad cow disease”)

– Lyme disease

• Detect presence of protein in a mixture of other proteins

• Determine size and test for post-translational modifications of a protein

• Analysis of protein-protein interactions

• Determine protein stability

• Structure domain analysis

Recomb DNA 96

49

Analysis of Protein Expression: Western Blot

• Total proteins isolated from tissues

• Separated by Poly‐Acrylamide Gel Electrophoresis (PAGE)

• Transferred to nitrocellulose filter

• Incubated with antibody against KLF15 (transcription factor)

• Different isoforms present in different tissues

Recomb DNA 98

Analysis of Protein Expression: Immuno-histochemistry / Immuno-fluorescence

• Purpose: Determine the cellular expression of protein within a tissue

• Tissue: Frozen or paraffin sections

• Probe: Antibodies that recognize specific protein (primary antibody)

• Detection: Tagged antibodies that recognize primary antibody (secondaryantibody)

– Immunohistochemistry: secondary tagged with enzyme; apply substrate for colorimetric detection.

– Immunofluorescence: secondary antibody tagged with fluorescent molecule

Section from fish retina:Stained for 2 different proteins using immuno-fluorescence (red) and immunohistochemistry (brown)

50

Analysis of Gene Function

Expression Vectors (plasmid, virus)Cloned genes are transcribed and translated into protein product

Gene is cloned under control of a strong promoter to drive high levels of expression.• Protein purification/biochemistry• Protein‐Protein interactions• Protein‐DNA interactions• Enzymatic activity

Transgenic animal production (more on this next semester)• Knock‐out mouse: eliminate gene to determine effect of lossof function on normal development/function

• Transgenic Animals: overexpress gene / force expression andanalyze effects

Transgenic Technology Transgenic

general term for genetically modified animal;

typically refers to random gene insertion

Knock-out

transgenic animal with a specific gene deleted/altered in the genome

Knock-in

transgenic animal designed to replace a target gene and with foreign gene

Conditional Knockout

transgenic animal that is engineered to delete/alter a target gene only under certain conditions

John Wilsonhttp://www.bcm.edu/biochem/?PMID=3946

http://www.fao.org/fishery/topic/14796/en

http://www.tetra-fish.com/glofish.aspx

51

Why Create Transgenic Animals?

To produce therapeutic proteins

e.g. Transgenic sheep and goats to produce human

proteins in their milk

insulin production

Research tools

Develop models for studying disease

e.g. Lumican knockouts: study corneal structure

MMP‐9 knockouts to study dry eye disease

Rhodopsin knock‐ins to model Retinitis pigmentosa

Replace normal genes with mutated genes to

determine consequences of mutation

Recomb DNA 102

Creating a Transgenic Mouse

Direct injection of DNAcontaining gene of interest into male pro-nucleus of mouse zygote of black mouse

Transplant into surrogate mother with different coat color (albino or brown)

Recover pups with black fur, check for presence of transgene (PCR; Southern)

• Not targeted: Insertion sites are random

• Transgene is typically inserted as multiple end to end copies

• Can induce mutations in unknown genes

• Does not remove existing gene

52

Recomb DNA 103

Generating Targeted Gene Knock-out

Replace original gene with engineered gene • homologous recombination is a rare event• much more difficult, time consuming• can take up to a year or more until you have mice to analyze

Uses cellular DNA repair mechanisms to replace endogenous gene with the engineered targeting construct

Transfect DNA construct into 108 embryonic stem cells

• 5-30 % of cells don’t take up any DNA

• In cells that take up the DNA the gene is not replaced:

• Most frequent: transgene DNA not incorporated into genome

• Others: transgene is randomly insertions

• ~1 cell in 100 million will have homologous recombination event(so, for 1 experiment you may only recover ~10 cells!)

Recomb DNA 104http://sgugenetics.pbworks.com/w/page/24704977/Creation%20of%20a%20%22Knockout%20Mouse%22

1. Embryonic Stem cells (ES cells) fromblasocyst of brown mouse

2. Clone targeting construct• contains mouse DNA from targeted region• engineered DNA containing desired

mutation/insert• genes for drug selection =

HSV-tk (thymidine kinase)neoR (neomycin resistance)

53

Recomb DNA 105

3. Introduce targeting construct into ES cells

4. Possible outcomes:• no DNA in cell frequent not useful• DNA randomly inserted moderate frequency not useful

• homologous recombination: normal gene replaced with targetingconstruct

this is a RARE event, but is the desired result

5. Use drug selection to obtain the rare ES cell with the correctly targeted gene:

• Cells with any of the engineered DNA construct will have neoR and survive

• Only cells with homologous recombination will lack HSV-TK and survive


6. Inject ES cells with targeted gene (from brown mouse) intoblastocysts from white mouse

7. Transplant into female host

8. Pups will be chimeras (brown and white)

54

Recomb DNA 107

Chimeras contain mixture of cells with and without transgene

Somatic cells containing transgene: not passed to next generation

Germline cells containing transgene: passed to next generation

Chimeras

Mythical Chimera

Chimeric mouse

chimera.clubhertzog.com/images/chimera.png

http://intramural.nimh.nih.gov/tgc/photogallery.html


Breed chimeras to normal mice

Genotype pups to identify mice with targeted gene

Breed to generate homozygous mice

Analyze effects of gene knockout or gene replacement on survival, development, pathology, etc.

Basic Molecular Approaches: Cloning and Beyond...1 Basic Molecular Approaches: Cloning and Beyond...

Documents

Transcript of Basic Molecular Approaches: Cloning and Beyond...1 Basic Molecular Approaches: Cloning and Beyond...