How do you identify and clone a gene of interest? Shotgun approach? Is there a better way?

How do you identify and clone a gene of interest?

• Shotgun approach?

• Is there a better way?

DNA Libraries

• What is a DNA library?• 2 main types• Genomic Library• cDNA Library

Genomic Library

• Random digestion of genome by restriction enzyme

• Fragments are cloned into a vector

• Recombinant vectors used to transform bacteria

• Disadvantages• Non-coding regions (introns) of

DNA• Needle in a haystack

cDNA Library• Double stranded DNA is

made from mRNA• Reverse Transcriptase• DNA polymerase

• Linker sequences added• Contain restriction sites

• Cloned into bacteria• Advantages• No introns, actively

expressed genes• Need abundant amount of

mRNA

Library Screening

• Now that you have a library, how do you find the gene of interest?• Colony Hybridization

Colony Hybridization

• Transformed bacteria are grown on an agar plate

• Cells are transferred to membrane

• Membrane is probed with radioactive oligonucleotide

Colony Hybridization

• Autoradiography will show which colonies contain the gene of interest

• The colony can be chosen and grown to isolate the plasmid

Cloning by PCR

What can you do with a cloned gene?

• Restriction Mapping• DNA sequencing (we’ll cover later)• Chromosomal location and copy number• FISH

• Southern Blotting• Study gene expression

Fluorescence in situ Hybridization

What can you do with a cloned gene?

• Restriction Mapping• DNA sequencing (we’ll cover later)• Chromosomal location and copy number• FISH

• Southern Blotting• Study gene expression

Studying Gene Expression

• Northern Blotting• Reverse Transcription PCR (RT-PCR)• Real-time PCR (qPCR)• In situ hybridization• DNA Microarray (we’ll discuss later)• Protein expression and purification• Gene mutagenesis studies• RNA Interference (RNAi)

Northern Blotting

• Similar to Southern blot• Use probes to detect

RNA• Can be used to study

mRNA expression in different tissues

Reverse Transcription PCR

• Useful with small amounts of RNA• RNA cannot be amplified by PCR• Use reverse transcriptase to make

double stranded cDNA• Primers are used to amplify the cDNA• Run on a gel• Allows to see expression patterns

Real-time PCR

• Aka quantitative PCR (qPCR)• Allows researcher to quantify amplification

reactions in real time• Requires expensive thermocycler• Reaction tube contains dye that glows from a

laser when bound to double stranded DNA• SYBR Green or TaqMan probes

• No need to run a gel

Real-time PCR

in situ Hybridization

• Use fluorescent probes to locate gene of interest in a tissue

Protein Expression and Purification

• Use transformed bacteria to produce protein from gene of interest

• More later

Gene Mutagenesis Studies

• aka site directed mutagenesis• Mutations created at specific nucleotides• Express in cells to see what happens

RNA Interference

(RNAi)

Applications of DNA Technology

The Human Genome Project

• Goals• Analyze genetic variations among humans• Map and sequence genomes of model

organisms• Develop new lab technologies• Disseminate information about the genome• Consider ethical, legal, and social issues of

genetic research

The Human Genome Project