Using mutants to clone genes

Size: px
Start display at page:

Download "Using mutants to clone genes"

Transcription

1 Using mutants to clone genes Objectives 1. What is positional cloning? 2. What is insertional tagging? 3. How can one confirm that the gene cloned is the same one that is mutated to give the phenotype of interest?

2 Reading References: Westhoff et. al. Molecular Plant Development: from gene to plant. Chapter 3:

3 Positional (map-based) Cloning Map based cloning is a labour-intensive but dependable method of cloning a gene using a mutant phenotype, RFLP-like genetic markers and genetic recombination. This method is generally restricted to organisms where the necessary tools, RFLP genetic map, physical map and or sequence of the genome are available.

4 Positional (map-based) Cloning 1. Use the mutant phenotype and DNA-based genetic markers of known position to map, using recombination, the gene of interest to a site on a specific chromosome.

5 DNA-Based Genetic Markers The genomes of two individuals of the same species are rarely identical and can have many nucleotide differences between them. These variations in DNA sequences often do not alter the function of a gene but can be used as phenotypes in genetic mapping by detecting the differences using: 1. restriction endonuclease digestion /DNA probes (restriction fragment-length polymorpism = RFLP) 2. PCR amplification (simple sequence-length polymorphism = SSLP) 3. a combination of both PCR amplification followed by restriction endonuclease digestion (cleaved amplified polymorphic sequences = CAPS).

6 Chromosome of Individual #1 Chromosome of Individual #2 CTGGGAATTCTTACC EcoR1 site Amplify by PCR CTGGGAAGTCTTACC Ind #1 Ind #2 #1 x #2 F1 Restrict amplified fragments with EcoR1 and separate on an electrophoretic gel.

7 Mapping to DNA-Based Genetic Markers The genomes of different varieties of Arabidopsis (ecotypes) differ in a large number of nucleotides. When these two varieties are crossed all the differences will segregate in the F2 progeny and can be mapped relative to one another or any novel phenotype in one of the parents. Commonly used Arabidopsis ecotypes are: Columbia (Col) Landsberg erecta (Ler)

8 Chromosome of Columbia ecotype with crl1-1 mutation crl1-1 CAPS16 C CAPS83 C CAPS8 C CAPS134 C CAPS41 C Chromosome of Landsberg erecta CAPS16 L CAPS83 L CRL1 CAPS8 L CAPS134 L CAPS41 L The crl-1 mutant phenotype is found to segregate with CAPS83 C and CAPS8 C but no others. Therefore the CRL1 gene must lie between these two CAPS sites.

9 Positional (map-based) Cloning 1. Use the mutant phenotype and DNA-based genetic markers to map, using recombination, the gene of interest to a region on a specific chromosome. 2. Examine the sequence of chromosomal DNA from that region to determine the number of annotated genes.

10 Arabidosis Genome

11 Arabidopsis Chromosome 1, UFO locus markers BACs Annotated genes

12 AT1G sequence Sequence: AT1G Date last modified Name AT1G Tair Accession Sequence: GenBank Accession NM_102835Sequence Length (bp) 1314 Sequence 1 ATGAAAGCTT TTAGATCTCT ACGTATACTA ATTTCCATCT CACGAACGAC 51 GACGAAGACA ACACCTCGTA ATCCCCATCA AGCACAAAAC TTTCTCCGCC 101 GATTTTACTC AGCGCAGCCG AATCTAGACG AACCCACTTC CATCAATGAA 151 GACGGATCAA GCAGCGACTC TGTTTTCGAT AGTAGTCAAT ACCCAATCGA 201 CGATTCCAAT GTAGATTCCG TGAAGAAGCC CAAGGAAGCA ACTTGGGATA 251 AAGGGTACAG AGAAAGAGTA AACAAAGCCT TCTTTGGAAA CTTGACAGAG 301 AAAGGTAAAG TGAAAGTTGC AGAAGAAGAG AGTTCTGAAG ATGATGAGGA 351 TAGTGTTGAT AGGTCAAGGA TTCTCGCTAA GGCTCTCTTA GAGGCTGCGT 401 TAGAGTCACC AGATGAAGAA CTTGGTGAAG GTGAAGTTAG AGAAGAAGAT 451 CAGAAGTCGC TTAATGTCGG CATCATCGGT CCACCTAATG CAGGAAAATC 501 TTCGCTGACT AATTTCATGG TTGGAACAAA GGTTGCTGCT GCTTCACGGA 551 AGACTAACAC GACGACACAT GAAGTGTTAG GAGTATTGAC AAAAGGAGAT 601 ACACAAGTCT GTTTCTTCGA TACTCCGGGT CTGATGCTGA AGAAAAGCGG 651 ATATGGTTAC AAAGACATCA AGGCTCGTGT GCAAAATGCT TGGACTTCTG 701 TTGACCTGTT TGATGTCCTC ATTGTTATGT TTGATGTCCA TAGGCATCTC 751 ATGAGTCCCG ATTCAAGAGT GGTACGCTTG ATCAAATACA TGGGAGAAGA 801 AGAAAATCCG AAACAAAAGC GCGTTTTATG TATGAACAAA GTTGATCTGG 851 TTGAGAAGAA AAAGGATCTA TTAAAGGTTG CTGAGGAGTT CCAAGATCTT 901 CCGGCATATG AAAGATACTT CATGATATCG GGACTTAAGG GATCAGGAGT 951 GAAAGATCTT TCCCAATACT TAATGGATCA GGCTGTTAAA AAACCATGGG 1001 AAGAAGATGC ATTCACGATG AGTGAAGAAG TCTTGAAGAA CATTTCTCTT 1051 GAAGTTGTTA GGGAGAGATT ACTAGACCAT GTCCATCAGG AAATACCATA 1101 TGGTCTGGAG CACCGTCTAG TGGACTGGAA AGAGCTGCGT GACGGGTCTC 1151 TTAGAATTGA ACAGCATCTC ATCACTCCTA AACTTAGCCA ACGCAAGATT 1201 CTTGTAGGCA AGGGCGGTTG CAAGATCGGG AGGATAGGAA TTGAGGCCAA 1251 TGAAGAACTC AGGAGAATAA TGAACCGCAA AGTTCATCTC ATTCTCCAGG 1301 TTAAGCTCAA GTGA Comments (shows only the most recent comments by default) Attribution type name datesubmitted_by AGI-TIGR submitted_by GenBank General comments or questions: curator@arabidopsis.org Seed or DNA stock questions (donations, availability, orders, etc): abrc@arabidopsis.org

13 Arabidopsis Chromosome 1, UFO locus markers ( ) BACs Annotated genes

14 Positional (map-based) Cloning 1. Use the mutant phenotype and DNA-based genetic markers to map, using recombination, the gene of interest to a region on a specific chromosome. 2. Examine the sequence of chromosomal DNA from that region to determine the number of annotated genes. 3. Narrow down to correct gene using predicted function, mutant allele sequence, complementation, expression analysis etc.

15 Insertional Tagging 1. Isolate mutant phenotype of interest from an insertional mutagenized population of plants. (Insertion DNA must be cloned: eg TDNA or Transposon). 2. Check that the transposon or TDNA in the mutant segregates with the mutant phenotype. ---The segregation of an insert can often be followed using the phenotype of a gene encoded in the insert (eg Kanamycin resistance), a probe for the insert or PCR primers that can amplify part of the insert. ---repetitive elements (eg. transposons) may complicate such an analysis.

16 Insertional Tagging 1. Isolate mutant phenotype of interest from an insertional mutagenized population of plants. (Insertion DNA must be cloned: eg TDNA or Transposon). 2. Check that the transposon or TDNA in the mutant segregates with the mutant phenotype. 3. Clone or amplify the chromosomal DNA at the site of insertion using the known sequence of the TDNA or transposon.

17 Insertional Tagging P coding region Gene X P coding region Gene X with insert Chromosome with genes including the one with insert

18 Insertional Tagging Digest genomic DNA with restriction endonuclease Identify the fragment carrying the insert: Eg. 1. Make a library and probe with the insertion sequences. 2. Ligate the DNA into circles and amplify using divergent insert primers (inverse PCR)

19 Inverse PCR ligate Amplify by PCR Clone into vector

20 Insertional Tagging 1. Isolate mutant phenotype of interest from an insertional mutagenized population of plants. (Insertion DNA must be cloned: eg TDNA or Transposon). 2. Check that the transposon or TDNA in the mutant segregates with the mutant phenotype. 3. Clone or amplify the chromosomal DNA at the site of insertion using the known sequence of the TDNA or transposon. 4. Sequence the DNA flanking the TDNA or transposon from the mutant and use the sequence to identify the wild type gene.

21 Insertional Tagging Clone into vector Use sequences from gene X to identify the wild type allele. P coding region Gene X

22 Connecting a cloned gene with a mutant phenotype Despite the method of cloning, one must confirm that the gene cloned (X) is the same gene that is mutated in mutant M (gene M). 1. Transgene complementation. The wild type fragment carrying gene X should be able to complement the recessive mutant M phenotype. This hypothesis can be tested by transforming the homozygous mutant with the wild type gene to check if it will restore the wild type phenotype. Eg. Transform pea rr plants with the SBEI gene to see if the gene will complement the mutant phenotype.

23 Connecting a cloned gene with a mutant phenotype 1. Transgene complementation. 2. Sequence gene X from several mutants homozygous for different alleles of gene M. If the M gene and X gene are the same then one should find a gene X mutation in every M mutant. This hypothesis can be tested by sequencing gene X from several M mutants each carrying a different allele of the gene of interest. Eg. Sequence the SBEI gene in several different r pea mutants each homozygous for a different r mutant allele. One should find a different mutation in the SBEI gene in every such r mutant.

24 Connecting a cloned gene with a mutant phenotype Genotype of plants homozygous for different alleles of the CRL1 gene: CRL1/CRL1 crl1-1/crl1-1 crl1-2/crl1-2 -cloned a wild type gene, MYB83, encoding a transcription factor. Is MYB83 gene CRL1? Clone MYB83 from each of the three plants above by PCR amplification. If MYB83 is CRL1 then MYB83 from CRL1/CRL1 will have a wild type sequence. MYB83 from crl1-1/crl1-1 will have a mutation. MYB83 from crl1-2/crl1-2 will also have a mutation but different from that of crl1-1.

25 Connecting a cloned gene with a mutant phenotype 1. Transgene complementation. 2. Sequence gene X from several mutants homozygous for different alleles of gene M. 3. Cosegregation analysis. DNA-based markers (RFLP) identifying gene X should cosegregate with the mutant phenotype M in genetic crosses. This hypothesis can be tested by crossing mutant M to a wild type plant, selffertilizing the F1 progeny to produce F2 progeny and scoring F2 plants for the mutant phenotype and the gene X molecular marker. Eg. Follow the segregation of an RFLP for the SBEI gene with the wrinkled seed phenotype of rr.

26 Connecting a cloned gene with a mutant phenotype 1. Transgene complementation. 2. Sequence gene X from several mutants homozygous for different alleles of gene M. 3. Cosegregation analysis. 4. Reverse genetics. Identify mutant alleles of gene X using reverse genetics. Mutations in gene X should have the same phenotype as mutant M and fail to complement the M mutant phenotype. Eg. A loss of function mutation in the SBEI gene should have a wrinkled seed phenotype.

Using mutants to clone genes

Using mutants to clone genes Using mutants to clone genes Objectives: 1. What is positional cloning? 2. What is insertional tagging? 3. How can one confirm that the gene cloned is the same one that is mutated to give the phenotype

More information

Lecture 2: Using Mutants to study Biological processes

Lecture 2: Using Mutants to study Biological processes Lecture 2: Using Mutants to study Biological processes Objectives: 1. Why use mutants? 2. How are mutants isolated? 3. What important genetic analyses must be done immediately after a genetic screen for

More information

Map-Based Cloning of Qualitative Plant Genes

Map-Based Cloning of Qualitative Plant Genes Map-Based Cloning of Qualitative Plant Genes Map-based cloning using the genetic relationship between a gene and a marker as the basis for beginning a search for a gene Chromosome walking moving toward

More information

Lecture 2-3: Using Mutants to study Biological processes

Lecture 2-3: Using Mutants to study Biological processes Lecture 2-3: Using Mutants to study Biological processes Objectives: 1. Why use mutants? 2. How are mutants isolated? 3. What important genetic analyses must be done immediately after a genetic screen

More information

Experimental Tools and Resources Available in Arabidopsis. Manish Raizada, University of Guelph, Canada

Experimental Tools and Resources Available in Arabidopsis. Manish Raizada, University of Guelph, Canada Experimental Tools and Resources Available in Arabidopsis Manish Raizada, University of Guelph, Canada Community website: The Arabidopsis Information Resource (TAIR) at http://www.arabidopsis.org Can order

More information

GENETICS EXAM 3 FALL a) is a technique that allows you to separate nucleic acids (DNA or RNA) by size.

GENETICS EXAM 3 FALL a) is a technique that allows you to separate nucleic acids (DNA or RNA) by size. Student Name: All questions are worth 5 pts. each. GENETICS EXAM 3 FALL 2004 1. a) is a technique that allows you to separate nucleic acids (DNA or RNA) by size. b) Name one of the materials (of the two

More information

The homeo'c gene AGAMOUS (AG) in Arabidopsis. Presenta'on by: Yang Liu and Zhonghang Zhang (Daisy) February 27th, 2018

The homeo'c gene AGAMOUS (AG) in Arabidopsis. Presenta'on by: Yang Liu and Zhonghang Zhang (Daisy) February 27th, 2018 The homeo'c gene AGAMOUS (AG) in Arabidopsis Presenta'on by: Yang Liu and Zhonghang Zhang (Daisy) February 27th, 2018 Arabidopsis Flower George Haughn, UBC Genes Direc'ng Flower Development in Arabidopsis

More information

R1 12 kb R1 4 kb R1. R1 10 kb R1 2 kb R1 4 kb R1

R1 12 kb R1 4 kb R1. R1 10 kb R1 2 kb R1 4 kb R1 Bcor101 Sample questions Midterm 3 1. The maps of the sites for restriction enzyme EcoR1 (R1) in the wild type and mutated cystic fibrosis genes are shown below: Wild Type R1 12 kb R1 4 kb R1 _ _ CF probe

More information

Chapter 8: Recombinant DNA. Ways this technology touches us. Overview. Genetic Engineering

Chapter 8: Recombinant DNA. Ways this technology touches us. Overview. Genetic Engineering Chapter 8 Recombinant DNA and Genetic Engineering Genetic manipulation Ways this technology touches us Criminal justice The Justice Project, started by law students to advocate for DNA testing of Death

More information

Abcam.com. hutton.ac.uk. Ipmdss.dk. Bo Gong and Eva Chou

Abcam.com. hutton.ac.uk. Ipmdss.dk. Bo Gong and Eva Chou Abcam.com Bo Gong and Eva Chou Ipmdss.dk hutton.ac.uk What is a homeotic gene? A gene which regulates the developmental fate of anatomical structures in an organism Why study them? Understand the underlying

More information

Applicazioni biotecnologiche

Applicazioni biotecnologiche Applicazioni biotecnologiche Analisi forense Sintesi di proteine ricombinanti Restriction Fragment Length Polymorphism (RFLP) Polymorphism (more fully genetic polymorphism) refers to the simultaneous occurrence

More information

1a. What is the ratio of feathered to unfeathered shanks in the offspring of the above cross?

1a. What is the ratio of feathered to unfeathered shanks in the offspring of the above cross? 1. Whether or not the shanks of chickens contains feathers is due to two independently assorting genes. Individuals have unfeathered shanks when they are homozygous for recessive genes at two loci; the

More information

BIO 202 Midterm Exam Winter 2007

BIO 202 Midterm Exam Winter 2007 BIO 202 Midterm Exam Winter 2007 Mario Chevrette Lectures 10-14 : Question 1 (1 point) Which of the following statements is incorrect. a) In contrast to prokaryotic DNA, eukaryotic DNA contains many repetitive

More information

Authors: Vivek Sharma and Ram Kunwar

Authors: Vivek Sharma and Ram Kunwar Molecular markers types and applications A genetic marker is a gene or known DNA sequence on a chromosome that can be used to identify individuals or species. Why we need Molecular Markers There will be

More information

BIO 304 Fall 2000 Exam II Name: ID #: 1. Fill in the blank with the best answer from the provided word bank. (2 pts each)

BIO 304 Fall 2000 Exam II Name: ID #: 1. Fill in the blank with the best answer from the provided word bank. (2 pts each) 1. Fill in the blank with the best answer from the provided word bank. (2 pts each) incomplete dominance conditional mutation penetrance expressivity pleiotropy Southern blotting hybridization epistasis

More information

Biology 105: Introduction to Genetics PRACTICE FINAL EXAM Part I: Definitions. Homology: Reverse transcriptase. Allostery: cdna library

Biology 105: Introduction to Genetics PRACTICE FINAL EXAM Part I: Definitions. Homology: Reverse transcriptase. Allostery: cdna library Biology 105: Introduction to Genetics PRACTICE FINAL EXAM 2006 Part I: Definitions Homology: Reverse transcriptase Allostery: cdna library Transformation Part II Short Answer 1. Describe the reasons for

More information

1a. What is the ratio of feathered to unfeathered shanks in the offspring of the above cross?

1a. What is the ratio of feathered to unfeathered shanks in the offspring of the above cross? Problem Set 5 answers 1. Whether or not the shanks of chickens contains feathers is due to two independently assorting genes. Individuals have unfeathered shanks when they are homozygous for recessive

More information

B) You can conclude that A 1 is identical by descent. Notice that A2 had to come from the father (and therefore, A1 is maternal in both cases).

B) You can conclude that A 1 is identical by descent. Notice that A2 had to come from the father (and therefore, A1 is maternal in both cases). Homework questions. Please provide your answers on a separate sheet. Examine the following pedigree. A 1,2 B 1,2 A 1,3 B 1,3 A 1,2 B 1,2 A 1,2 B 1,3 1. (1 point) The A 1 alleles in the two brothers are

More information

Concepts: What are RFLPs and how do they act like genetic marker loci?

Concepts: What are RFLPs and how do they act like genetic marker loci? Restriction Fragment Length Polymorphisms (RFLPs) -1 Readings: Griffiths et al: 7th Edition: Ch. 12 pp. 384-386; Ch.13 pp404-407 8th Edition: pp. 364-366 Assigned Problems: 8th Ch. 11: 32, 34, 38-39 7th

More information

Construction of plant complementation vector and generation of transgenic plants

Construction of plant complementation vector and generation of transgenic plants MATERIAL S AND METHODS Plant materials and growth conditions Arabidopsis ecotype Columbia (Col0) was used for this study. SALK_072009, SALK_076309, and SALK_027645 were obtained from the Arabidopsis Biological

More information

7 Gene Isolation and Analysis of Multiple

7 Gene Isolation and Analysis of Multiple Genetic Techniques for Biological Research Corinne A. Michels Copyright q 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic) 7 Gene Isolation and Analysis of Multiple

More information

Before starting, write your name on the top of each page Make sure you have all pages

Before starting, write your name on the top of each page Make sure you have all pages Biology 105: Introduction to Genetics Name Student ID Before starting, write your name on the top of each page Make sure you have all pages You can use the back-side of the pages for scratch, but we will

More information

Midterm 1 Results. Midterm 1 Akey/ Fields Median Number of Students. Exam Score

Midterm 1 Results. Midterm 1 Akey/ Fields Median Number of Students. Exam Score Midterm 1 Results 10 Midterm 1 Akey/ Fields Median - 69 8 Number of Students 6 4 2 0 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 Exam Score Quick review of where we left off Parental type: the

More information

Sept 2. Structure and Organization of Genomes. Today: Genetic and Physical Mapping. Sept 9. Forward and Reverse Genetics. Genetic and Physical Mapping

Sept 2. Structure and Organization of Genomes. Today: Genetic and Physical Mapping. Sept 9. Forward and Reverse Genetics. Genetic and Physical Mapping Sept 2. Structure and Organization of Genomes Today: Genetic and Physical Mapping Assignments: Gibson & Muse, pp.4-10 Brown, pp. 126-160 Olson et al., Science 245: 1434 New homework:due, before class,

More information

Molecular Biology (2)

Molecular Biology (2) Molecular Biology (2) Restriction endonucleases, RFLP, and gene cloning Mamoun Ahram, PhD Second semester, 2017-2018 Resources This lecture Cooper, pp 120-124 Endonucleases Enzymes that degrade DNA within

More information

Recombinant DNA Libraries and Forensics

Recombinant DNA Libraries and Forensics MIT Department of Biology 7.014 Introductory Biology, Spring 2005 A. Library construction Recombinant DNA Libraries and Forensics Recitation Section 18 Answer Key April 13-14, 2005 Recall that earlier

More information

Enzyme that uses RNA as a template to synthesize a complementary DNA

Enzyme that uses RNA as a template to synthesize a complementary DNA Biology 105: Introduction to Genetics PRACTICE FINAL EXAM 2006 Part I: Definitions Homology: Comparison of two or more protein or DNA sequence to ascertain similarities in sequences. If two genes have

More information

Lecture 12. Genomics. Mapping. Definition Species sequencing ESTs. Why? Types of mapping Markers p & Types

Lecture 12. Genomics. Mapping. Definition Species sequencing ESTs. Why? Types of mapping Markers p & Types Lecture 12 Reading Lecture 12: p. 335-338, 346-353 Lecture 13: p. 358-371 Genomics Definition Species sequencing ESTs Mapping Why? Types of mapping Markers p.335-338 & 346-353 Types 222 omics Interpreting

More information

Introduction to some aspects of molecular genetics

Introduction to some aspects of molecular genetics Introduction to some aspects of molecular genetics Julius van der Werf (partly based on notes from Margaret Katz) University of New England, Armidale, Australia Genetic and Physical maps of the genome...

More information

I.1 The Principle: Identification and Application of Molecular Markers

I.1 The Principle: Identification and Application of Molecular Markers I.1 The Principle: Identification and Application of Molecular Markers P. Langridge and K. Chalmers 1 1 Introduction Plant breeding is based around the identification and utilisation of genetic variation.

More information

BSCI410-Liu/Spring 06 Exam #1 Feb. 23, 06

BSCI410-Liu/Spring 06 Exam #1 Feb. 23, 06 Your Name: Your UID# 1. (20 points) Match following mutations with corresponding mutagens (X-RAY, Ds transposon excision, UV, EMS, Proflavin) a) Thymidine dimmers b) Breakage of DNA backbone c) Frameshift

More information

Molecular Cell Biology - Problem Drill 11: Recombinant DNA

Molecular Cell Biology - Problem Drill 11: Recombinant DNA Molecular Cell Biology - Problem Drill 11: Recombinant DNA Question No. 1 of 10 1. Which of the following statements about the sources of DNA used for molecular cloning is correct? Question #1 (A) cdna

More information

Chapter 20 Recombinant DNA Technology. Copyright 2009 Pearson Education, Inc.

Chapter 20 Recombinant DNA Technology. Copyright 2009 Pearson Education, Inc. Chapter 20 Recombinant DNA Technology Copyright 2009 Pearson Education, Inc. 20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction Enzymes and DNA Cloning Vectors Recombinant DNA refers

More information

Biotechnology Chapter 20

Biotechnology Chapter 20 Biotechnology Chapter 20 DNA Cloning DNA Cloning AKA Plasmid-based transformation or molecular cloning First off-let s sum up what happens. A plasmid is taken from a bacteria A gene is inserted into the

More information

Trasposable elements: Uses of P elements Problem set B at the end

Trasposable elements: Uses of P elements Problem set B at the end Trasposable elements: Uses of P elements Problem set B at the end P-elements have revolutionized the way Drosophila geneticists conduct their research. Here, we will discuss just a few of the approaches

More information

Genetics Lecture Notes Lectures 13 16

Genetics Lecture Notes Lectures 13 16 Genetics Lecture Notes 7.03 2005 Lectures 13 16 Lecture 13 Transposable elements Transposons are usually from 10 3 to 10 4 base pairs in length, depending on the transposon type. The key property of transposons

More information

RFLP Method - Restriction Fragment Length Polymorphism

RFLP Method - Restriction Fragment Length Polymorphism RFLP Method - Restriction Fragment Length Polymorphism RFLP (often pronounced "rif lip", as if it were a word) is a method used by molecular biologists to follow a particular sequence of DNA as it is passed

More information

Restriction Enzymes (endonucleases)

Restriction Enzymes (endonucleases) In order to understand and eventually manipulate DNA (human or otherwise) an array of DNA technologies have been developed. Here are some of the tools: Restriction Enzymes (endonucleases) In order to manipulate

More information

Restriction Site Mapping:

Restriction Site Mapping: Restriction Site Mapping: In making genomic library the DNA is cut with rare cutting enzymes and large fragments of the size of 100,000 to 1000, 000bp. They are ligated to vectors such as Pacmid or YAC

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION doi:10.1038/nature09937 a Name Position Primersets 1a 1b 2 3 4 b2 Phenotype Genotype b Primerset 1a D T C R I E 10000 8000 6000 5000 4000 3000 2500 2000 1500 1000 800 Donor (D)

More information

BSCI410-Liu/Spring 09/Feb 26 Exam #1 Your name:

BSCI410-Liu/Spring 09/Feb 26 Exam #1 Your name: 1. (20 points) Give the name of a mutagen that could cause the following damages to DNA: a) Thymidine dimers UV b) Breakage of DNA backbone X-Ray c) 2 bp insertion (frameshift mutation) proflavin, acridine

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Gene replacements and insertions in rice by intron targeting using CRISPR Cas9 Table of Contents Supplementary Figure 1. sgrna-induced targeted mutations in the OsEPSPS gene in rice protoplasts. Supplementary

More information

BIOLOGY - CLUTCH CH.20 - BIOTECHNOLOGY.

BIOLOGY - CLUTCH CH.20 - BIOTECHNOLOGY. !! www.clutchprep.com CONCEPT: DNA CLONING DNA cloning is a technique that inserts a foreign gene into a living host to replicate the gene and produce gene products. Transformation the process by which

More information

Chapter 5. Structural Genomics

Chapter 5. Structural Genomics Chapter 5. Structural Genomics Contents 5. Structural Genomics 5.1. DNA Sequencing Strategies 5.1.1. Map-based Strategies 5.1.2. Whole Genome Shotgun Sequencing 5.2. Genome Annotation 5.2.1. Using Bioinformatic

More information

A) (5 points) As the starting step isolate genomic DNA from

A) (5 points) As the starting step isolate genomic DNA from GS Final Exam Spring 00 NAME. bub ts is a recessive temperature sensitive mutation in yeast. At º C bub ts cells grow normally, but at º C they die. Use the information below to clone the wild-type BUB

More information

SUPPLEMENTARY RESEARCH DATA

SUPPLEMENTARY RESEARCH DATA SUPPLEMENTARY RESEARCH DATA Supplementary Experimental Procedures Small RNA Analysis RNA was isolated using the mirvana mirna isolation kit (Ambion). Six µg of RNA was resolved by electrophoresis using

More information

Molecular Genetics Techniques. BIT 220 Chapter 20

Molecular Genetics Techniques. BIT 220 Chapter 20 Molecular Genetics Techniques BIT 220 Chapter 20 What is Cloning? Recombinant DNA technologies 1. Producing Recombinant DNA molecule Incorporate gene of interest into plasmid (cloning vector) 2. Recombinant

More information

Erhard et al. (2013). Plant Cell /tpc

Erhard et al. (2013). Plant Cell /tpc Supplemental Figure 1. c1-hbr allele structure. Diagram of the c1-hbr allele found in stocks segregating 1:1 for rpd1-1 and rpd1-2 homozygous mutants showing the presence of a 363 base pair (bp) Heartbreaker

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:10.1038/nature09861 & &' -(' ()*+ ')(+,,(','-*+,&,,+ ',+' ' 23,45/0*6787*9:./09 ;78?4?@*+A786?B- &' )*+*(,-* -(' ()*+ ')(+,,(','-*+,&,,+ ',+'./)*+*(,-*..)*+*(,-*./)*+*(,-*.0)*+*(,-*..)*+*(,-*

More information

PLNT3140 INTRODUCTORY CYTOGENETICS FINAL EXAMINATION December 6, 2008 Time: 1:30-3:30 pm Location: E2-150 EIT Complex, seats 1-18

PLNT3140 INTRODUCTORY CYTOGENETICS FINAL EXAMINATION December 6, 2008 Time: 1:30-3:30 pm Location: E2-150 EIT Complex, seats 1-18 PLNT3140 INTRODUCTORY CYTOGENETICS FINAL EXAMINATION December 6, 2008 Time: 1:30-3:30 pm Location: E2-150 EIT Complex, seats 1-18 Answer any combination of questions totalling to exactly 100 points. (There

More information

Genetics - Problem Drill 19: Dissection of Gene Function: Mutational Analysis of Model Organisms

Genetics - Problem Drill 19: Dissection of Gene Function: Mutational Analysis of Model Organisms Genetics - Problem Drill 19: Dissection of Gene Function: Mutational Analysis of Model Organisms No. 1 of 10 1. The mouse gene knockout is based on. (A) Homologous recombination (B) Site-specific recombination

More information

Recitation CHAPTER 9 DNA Technologies

Recitation CHAPTER 9 DNA Technologies Recitation CHAPTER 9 DNA Technologies DNA Cloning: General Scheme A cloning vector and eukaryotic chromosomes are separately cleaved with the same restriction endonuclease. (A single chromosome is shown

More information

Chapter 6 - Molecular Genetic Techniques

Chapter 6 - Molecular Genetic Techniques Chapter 6 - Molecular Genetic Techniques Two objects of molecular & genetic technologies For analysis For generation Molecular genetic technologies! For analysis DNA gel electrophoresis Southern blotting

More information

Genetic and Molecular Characterization of Host-Plant Resistance to Root-knot Nematodes and Fusarium Wilt in Cotton

Genetic and Molecular Characterization of Host-Plant Resistance to Root-knot Nematodes and Fusarium Wilt in Cotton Genetic and Molecular Characterization of Host-Plant Resistance to Root-knot Nematodes and Fusarium Wilt in Cotton Phil Roberts & Congli Wang, Department of Nematology Univ. of California, Riverside Project

More information

Test Bank for Molecular Cell Biology 7th Edition by Lodish

Test Bank for Molecular Cell Biology 7th Edition by Lodish Test Bank for Molecular Cell Biology 7th Edition by Lodish Link download full: http://testbankair.com/download/test-bank-formolecular-cell-biology-7th-edition-by-lodish/ Chapter 5 Molecular Genetic Techniques

More information

7.012 Problem Set 5. Question 1

7.012 Problem Set 5. Question 1 Name Section 7.012 Problem Set 5 Question 1 While studying the problem of infertility, you attempt to isolate a hypothetical rabbit gene that accounts for the prolific reproduction of rabbits. After much

More information

3 Designing Primers for Site-Directed Mutagenesis

3 Designing Primers for Site-Directed Mutagenesis 3 Designing Primers for Site-Directed Mutagenesis 3.1 Learning Objectives During the next two labs you will learn the basics of site-directed mutagenesis: you will design primers for the mutants you designed

More information

Microsatellite markers

Microsatellite markers Microsatellite markers Review of repetitive sequences 25% 45% 8% 21% 13% 3% Mobile genetic elements: = dispersed repeat included: transposition: moving in the form of DNA by element coding for transposases.

More information

IDN1 and IDN2: two proteins required for de novo DNA methylation in Arabidopsis thaliana.

IDN1 and IDN2: two proteins required for de novo DNA methylation in Arabidopsis thaliana. IDN1 and IDN2: two proteins required for de novo DNA methylation in Arabidopsis thaliana. Israel Ausin, Todd C. Mockler, Joanne Chory, and Steven E. Jacobsen Col Ler nrpd1a rdr2 dcl3-1 ago4-1 idn1-1 idn2-1

More information

INTERNATIONAL UNION FOR THE PROTECTION OF NEW VARIETIES OF PLANTS GENEVA

INTERNATIONAL UNION FOR THE PROTECTION OF NEW VARIETIES OF PLANTS GENEVA E BMT Guidelines (proj.4) ORIGINAL: English DATE: December 21, 2005 INTERNATIONAL UNION FOR THE PROTECTION OF NEW VARIETIES OF PLANTS GENEVA GUIDELINES FOR DNA-PROFILING: MOLECULAR MARKER SELECTION AND

More information

Biology 163 Laboratory in Genetics, Final Exam, Dec. 10, 2005

Biology 163 Laboratory in Genetics, Final Exam, Dec. 10, 2005 1 Biology 163 Laboratory in Genetics, Final Exam, Dec. 10, 2005 Honor Pledge: I have neither given nor received any unauthorized help on this exam: Name Printed: Signature: 1. (2 pts) If you see the following

More information

Your name: BSCI410-LIU/Spring 2007 Homework #2 Due March 27 (Tu), 07

Your name: BSCI410-LIU/Spring 2007 Homework #2 Due March 27 (Tu), 07 BSCI410-LIU/Spring 2007 Homework #2 Due March 27 (Tu), 07 KEY 1. What are each of the following molecular markers? (Indicate (a) what they stand for; (b) the nature of the molecular polymorphism and (c)

More information

PCB Fa Falll l2012

PCB Fa Falll l2012 PCB 5065 Fall 2012 Molecular Markers Bassi and Monet (2008) Morphological Markers Cai et al. (2010) JoVE Cytogenetic Markers Boskovic and Tobutt, 1998 Isozyme Markers What Makes a Good DNA Marker? High

More information

Genome research in eukaryotes

Genome research in eukaryotes Functional Genomics Genome and EST sequencing can tell us how many POTENTIAL genes are present in the genome Proteomics can tell us about proteins and their interactions The goal of functional genomics

More information

(a) (3 points) Which of these plants (use number) show e/e pattern? Which show E/E Pattern and which showed heterozygous e/e pattern?

(a) (3 points) Which of these plants (use number) show e/e pattern? Which show E/E Pattern and which showed heterozygous e/e pattern? 1. (20 points) What are each of the following molecular markers? (Indicate (a) what they stand for; (b) the nature of the molecular polymorphism and (c) Methods of detection (such as gel electrophoresis,

More information

Chapter 10 Genetic Engineering: A Revolution in Molecular Biology

Chapter 10 Genetic Engineering: A Revolution in Molecular Biology Chapter 10 Genetic Engineering: A Revolution in Molecular Biology Genetic Engineering Direct, deliberate modification of an organism s genome bioengineering Biotechnology use of an organism s biochemical

More information

MCB 102 University of California, Berkeley August 11 13, Problem Set 8

MCB 102 University of California, Berkeley August 11 13, Problem Set 8 MCB 102 University of California, Berkeley August 11 13, 2009 Isabelle Philipp Handout Problem Set 8 The answer key will be posted by Tuesday August 11. Try to solve the problem sets always first without

More information

Proposed experiments to study SAR

Proposed experiments to study SAR Proposed experiments to study SAR Question or hypothesis Proposed experiments Expectation 1. Arabidopsis can be used to study SAR Test SAR response in Arabidopsis Yes, SAR on Arabidopsis 2. What are contributing

More information

New methodologies for gene. identification and characterization: Concepts and case studies

New methodologies for gene. identification and characterization: Concepts and case studies Corso di Formazione GenHORT New methodologies for gene identification and characterization: Concepts and case studies 24 Marzo 2014 Federica Consiglio Giorgia Batelli Michael Van Oosten Course Outline

More information

BENG 183 Trey Ideker. Genotyping. To be covered in one 1.5 hr lecture

BENG 183 Trey Ideker. Genotyping. To be covered in one 1.5 hr lecture BENG 183 Trey Ideker Genotyping To be covered in one 1.5 hr lecture Genetic variation: Some basic definitions Allele Alternative form of a genetic locus inherited separately from each parent Polymorphism

More information

Q1 (1 point): Explain why a lettuce leaf wilts when it is placed in a concentrated salt solution.

Q1 (1 point): Explain why a lettuce leaf wilts when it is placed in a concentrated salt solution. Short questions 1 point per question. Q1 (1 point): Explain why a lettuce leaf wilts when it is placed in a concentrated salt solution. Q2 (1 point): Put a cross by the correct answer(s) below. The Na

More information

Phenotype analysis: biological-biochemical analysis. Genotype analysis: molecular and physical analysis

Phenotype analysis: biological-biochemical analysis. Genotype analysis: molecular and physical analysis 1 Genetic Analysis Phenotype analysis: biological-biochemical analysis Behaviour under specific environmental conditions Behaviour of specific genetic configurations Behaviour of progeny in crosses - Genotype

More information

Marker types. Potato Association of America Frederiction August 9, Allen Van Deynze

Marker types. Potato Association of America Frederiction August 9, Allen Van Deynze Marker types Potato Association of America Frederiction August 9, 2009 Allen Van Deynze Use of DNA Markers in Breeding Germplasm Analysis Fingerprinting of germplasm Arrangement of diversity (clustering,

More information

Q1 (1 point): Explain why a lettuce leaf wilts when it is placed in a concentrated salt solution.

Q1 (1 point): Explain why a lettuce leaf wilts when it is placed in a concentrated salt solution. Short questions 1 point per question. Q1 (1 point): Explain why a lettuce leaf wilts when it is placed in a concentrated salt solution. Answer: Water is sucked out of the cells by osmosis (this reduces

More information

A Versatile and Highly Reliable Method of Identifying Genes Responsible for Mutations Caused by Highly Active Transposable Elements

A Versatile and Highly Reliable Method of Identifying Genes Responsible for Mutations Caused by Highly Active Transposable Elements JARQ 51 (1), 51-57 (2017) https://www.jircas.go.jp A Versatile and Highly Reliable Method of Identifying Genes Responsible for Mutations Caused by Highly Active Transposable Elements Takaaki NISHIJIMA

More information

Problem Set 4

Problem Set 4 7.016- Problem Set 4 Question 1 Arginine biosynthesis is an example of multi-step biochemical pathway where each step is catalyzed by a specific enzyme (E1, E2 and E3) as is outlined below. E1 E2 E3 A

More information

[Presented by: Andrew Howlett, Cruise Slater, Mahmud Hasan, Greg Dale]

[Presented by: Andrew Howlett, Cruise Slater, Mahmud Hasan, Greg Dale] Mutational Dissection [Presented by: Andrew Howlett, Cruise Slater, Mahmud Hasan, Greg Dale] Introduction What is the point of Mutational Dissection? It allows understanding of normal biological functions

More information

Supplemental Data. Zhou et al. (2016). Plant Cell /tpc

Supplemental Data. Zhou et al. (2016). Plant Cell /tpc Supplemental Figure 1. Confirmation of mutant mapping results. (A) Complementation assay with stably transformed genomic fragments (ComN-N) (2 kb upstream of TSS and 1.5 kb downstream of TES) and CaMV

More information

Mapping and Mapping Populations

Mapping and Mapping Populations Mapping and Mapping Populations Types of mapping populations F 2 o Two F 1 individuals are intermated Backcross o Cross of a recurrent parent to a F 1 Recombinant Inbred Lines (RILs; F 2 -derived lines)

More information

INTERNATIONAL UNION FOR THE PROTECTION OF NEW VARIETIES OF PLANTS

INTERNATIONAL UNION FOR THE PROTECTION OF NEW VARIETIES OF PLANTS ORIGINAL: English DATE: October 21, 2010 INTERNATIONAL UNION FOR THE PROTECTION OF NEW VARIETIES OF PLANTS GENEVA E GUIDELINES FOR DNA-PROFILING: MOLECULAR MARKER SELECTION AND DATABASE CONSTRUCTION (

More information

Phenotype analysis: biological-biochemical analysis. Genotype analysis: molecular and physical analysis

Phenotype analysis: biological-biochemical analysis. Genotype analysis: molecular and physical analysis 1 Genetic Analysis Phenotype analysis: biological-biochemical analysis Behaviour under specific environmental conditions Behaviour of specific genetic configurations Behaviour of progeny in crosses - Genotype

More information

MOLECULAR TYPING TECHNIQUES

MOLECULAR TYPING TECHNIQUES MOLECULAR TYPING TECHNIQUES RATIONALE Used for: Identify the origin of a nosocomial infection Identify transmission of disease between individuals Recognise emergence of a hypervirulent strain Recognise

More information

BA, BSc, and MSc Degree Examinations

BA, BSc, and MSc Degree Examinations Examination Candidate Number: Desk Number: BA, BSc, and MSc Degree Examinations 2017-8 Department : BIOLOGY Title of Exam: Genetics Time Allowed: 1 hour and 30 minutes Marking Scheme: Total marks available

More information

Biology 163 Laboratory in Genetics Midterm 2, Nov. 14, Honor Pledge: I have neither given nor received any unauthorized help on this exam:

Biology 163 Laboratory in Genetics Midterm 2, Nov. 14, Honor Pledge: I have neither given nor received any unauthorized help on this exam: 1 Biology 163 Laboratory in Genetics Midterm 2, Nov. 14, 2005 Honor Pledge: I have neither given nor received any unauthorized help on this exam: Name Printed: ignature: 1. Normally you need to cross two

More information

Chapter 5 Genetic Analysis in Cell Biology. (textbook: Molecular Cell Biology 6 ed, Lodish section: )

Chapter 5 Genetic Analysis in Cell Biology. (textbook: Molecular Cell Biology 6 ed, Lodish section: ) Chapter 5 Genetic Analysis in Cell Biology (textbook: Molecular Cell Biology 6 ed, Lodish section: 5.1+5.4-5.5) Understanding gene function: relating function, location, and structure of gene products

More information

http://fire.biol.wwu.edu/trent/trent/direct_detection_of_genotype.html 1 Like most other model organism Arabidopsis thaliana has a sequenced genome? What do we mean by sequenced genome? What sort of info

More information

Population Genetics. If we closely examine the individuals of a population, there is almost always PHENOTYPIC

Population Genetics. If we closely examine the individuals of a population, there is almost always PHENOTYPIC 1 Population Genetics How Much Genetic Variation exists in Natural Populations? Phenotypic Variation If we closely examine the individuals of a population, there is almost always PHENOTYPIC VARIATION -

More information

STANDARD CLONING PROCEDURES. Shotgun cloning (using a plasmid vector and E coli as a host).

STANDARD CLONING PROCEDURES. Shotgun cloning (using a plasmid vector and E coli as a host). STANDARD CLONING PROCEDURES Shotgun cloning (using a plasmid vector and E coli as a host). 1) Digest donor DNA and plasmid DNA with the same restriction endonuclease 2) Mix the fragments together and treat

More information

Next Generation Genetics: Using deep sequencing to connect phenotype to genotype

Next Generation Genetics: Using deep sequencing to connect phenotype to genotype Next Generation Genetics: Using deep sequencing to connect phenotype to genotype http://1001genomes.org Korbinian Schneeberger Connecting Genotype and Phenotype Genotyping SNPs small Resequencing SVs*

More information

Mos1 insertion. MosTIC protocol-11/2006. repair template - Mos1 transposase expression - Mos1 excision - DSB formation. homolog arm.

Mos1 insertion. MosTIC protocol-11/2006. repair template - Mos1 transposase expression - Mos1 excision - DSB formation. homolog arm. MosTIC (Mos1 excision induced Transgene Instructed gene Conversion) Valérie Robert (vrobert@biologie.ens.fr) and Jean-Louis Bessereau (jlbesse@biologie.ens.fr) (November 2006) Introduction: MosTIC (Robert

More information

2014 Pearson Education, Inc. CH 8: Recombinant DNA Technology

2014 Pearson Education, Inc. CH 8: Recombinant DNA Technology CH 8: Recombinant DNA Technology Biotechnology the use of microorganisms to make practical products Recombinant DNA = DNA from 2 different sources What is Recombinant DNA Technology? modifying genomes

More information

Biology 201 (Genetics) Exam #3 120 points 20 November Read the question carefully before answering. Think before you write.

Biology 201 (Genetics) Exam #3 120 points 20 November Read the question carefully before answering. Think before you write. Name KEY Section Biology 201 (Genetics) Exam #3 120 points 20 November 2006 Read the question carefully before answering. Think before you write. You will have up to 50 minutes to take this exam. After

More information

PIE1 ARP6 SWC6 KU70 ARP6 PIE1. HSA SNF2_N HELICc SANT. pie1-3 A1,A2 K1,K2 K1,K3 K3,LB2 A3, A4 A3,LB1 A1,A2 K1,K2 K1,K3. swc6-1 A3,A4.

PIE1 ARP6 SWC6 KU70 ARP6 PIE1. HSA SNF2_N HELICc SANT. pie1-3 A1,A2 K1,K2 K1,K3 K3,LB2 A3, A4 A3,LB1 A1,A2 K1,K2 K1,K3. swc6-1 A3,A4. A B N-terminal SWC2 H2A.Z SWC6 ARP6 PIE1 HSA SNF2_N HELICc SANT C pie1-3 D PIE1 ARP6 5 Kb A1 200 bp A3 A2 LB1 arp6-3 A4 E A1,A2 A3, A4 A3,LB1 K1,K2 K1,K3 K3,LB2 SWC6 swc6-1 A1,A2 A3,A4 K1,K2 K1,K3 100

More information

XXII DNA cloning and sequencing. Outline

XXII DNA cloning and sequencing. Outline XXII DNA cloning and sequencing 1) Deriving DNA for cloning Outline 2) Vectors; forming recombinant DNA; cloning DNA; and screening for clones containing recombinant DNA [replica plating and autoradiography;

More information

CH 8: Recombinant DNA Technology

CH 8: Recombinant DNA Technology CH 8: Recombinant DNA Technology Biotechnology the use of microorganisms to make practical products Recombinant DNA = DNA from 2 different sources What is Recombinant DNA Technology? modifying genomes

More information

B. Incorrect! Ligation is also a necessary step for cloning.

B. Incorrect! Ligation is also a necessary step for cloning. Genetics - Problem Drill 15: The Techniques in Molecular Genetics No. 1 of 10 1. Which of the following is not part of the normal process of cloning recombinant DNA in bacteria? (A) Restriction endonuclease

More information

CONSTRUCTION OF GENOMIC LIBRARY

CONSTRUCTION OF GENOMIC LIBRARY MODULE 4-LECTURE 4 CONSTRUCTION OF GENOMIC LIBRARY 4-4.1. Introduction A genomic library is an organism specific collection of DNA covering the entire genome of an organism. It contains all DNA sequences

More information

HC70AL SUMMER 2014 PROFESSOR BOB GOLDBERG Gene Annotation Worksheet

HC70AL SUMMER 2014 PROFESSOR BOB GOLDBERG Gene Annotation Worksheet HC70AL SUMMER 2014 PROFESSOR BOB GOLDBERG Gene Annotation Worksheet NAME: DATE: QUESTION ONE Using primers given to you by your TA, you carried out sequencing reactions to determine the identity of the

More information

Genetics Test. Multiple Choice Identify the choice that best completes the statement or answers the question.

Genetics Test. Multiple Choice Identify the choice that best completes the statement or answers the question. Genetics Test Multiple Choice Identify the choice that best completes the statement or answers the question. 41. Situations in which one allele for a gene is not completely dominant over another allele

More information

BC2004 Review Sheet for Lab Exercises 7-11 Spring Semester 2005

BC2004 Review Sheet for Lab Exercises 7-11 Spring Semester 2005 BC2004 Review Sheet for Lab Exercises 7-11 Spring Semester 2005 Lab Exercise 7 Drosophila crosses, three weeks Vocabulary: phenotype, genotype, gene, allele, locus (loci), sex chromosomes, autosomes, homozygous,

More information