recombinant dna and biotechnology. recombinant dna –cleaving and rejoining dna –getting new...

Recombinant DNA and Biotechnology

Recombinant DNA and Biotechnology

• Recombinant DNA– Cleaving and Rejoining DNA– Getting New Genes into Cells– Identifying and Cloning Genes for

• Biotechnology – Applications of DNA Manipulation

Cleaving DNA• restriction enzymes (REs)

– Bacterial defense against infection by phage– Enzymes that cut phosphodiester bonds of DNA

Examples

• EcoRI binds & cuts DNA at the following sequence:– 5 ... GAATTC ... 3– 3... CTTAAG ... 5

• The sequence is palindromic:– reads the same 5-to-3

on both strands.

Methylation protects bacterial chromosomal sites from digestion

Fragmentation of DNA by REs

• Using EcoRI on a long stretch of DNA would create fragments with an average length of 4,096 bases.

– Why??

• Using EcoRI to cut up small viral genomes may result in only a few fragments.

• For a eukaryotic genome with tens of millions of base pairs, the number of fragments will be very large.

Analysis of DNA Fragments

• Fragments of DNA can be separated based on their size using gel electrophoresis. – DNA is negatively charged - ionized phosphate groups– DNA molecules migrate toward a positive pole of an

electric field.• Agarose or Polyacrylamide Gels

– a porous matrix through which molecules can pass– speed of movement proportional to molecule’s size– Smaller molecules (fewer bp) migrate more easily

• After separation, the molecules must be stained to bee visualized– bound to a dye that fluoresces under UV light.

Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)

• Electrophoresis– Movement through a matrix driven by an electric field– Matrix restricts passage of molecules based on their size

Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)

Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 3)

Manipulating DNA Fragments

• Electrophoresis gives two types of information:– Size of the DNA (or RNA) fragments can be

determined by comparison to fragments of known size – A specific DNA (or RNA) sequence can be identified

using a labeled single-stranded nucleic acid probe complementary to the target being identified

• The specific fragment can be cut out as a lump of gel and removed by diffusion into a small volume of water.

Figure 16.3 Analyzing DNA Fragments

• Southern Blotting – identification of separated

DNA frags• Northern Blotting

– identification of separated RNA frags

Figure 16.4 Cutting and Splicing DNA

• Some REs leave staggered ends of single-stranded DNA, or “sticky” ends

• These ends can bind to the same sequence at the end of another EcoRI-cut fragment of DNA

Manipulating DNA & Genes

• Molecules of DNA can not be worked with individually, - only in large numbers (copies)

• Recombinant DNA allows the joining of any DNA sequence to any other

• This allows the joining of fragments of DNA to vectors that permit making many copies of the desired DNA fragment

• Production of many copies of a particular gene is called cloning

Cloning

• Bacterial plasmids and bacteriophage DNA are used as vectors

• Plasmids – – naturally occurring – small (few 1000 bp), circular DNA molecules– replicate autonomously from chromosome

• Phage –– inject & replicate their DNA in bacterial host– many copies of phage DNA made to be inserted into

phage progeny

Vectors

• Vectors have four characteristics:

– The ability to replicate independently in the host cell

– A recognition sequence for a restriction enzyme, permitting it to form recombinant DNA

– A reporter gene(s) that will allow for selection/identification of host cells containing it

– A small size in comparison to host chromosomes

Insertion of DNA into a Plasmid Vector

Using a Lamba () Phage Vector

Vectors• Plasmids

– 5-10Kbp inserts – Genomic DNA or cDNAs

• Phage– 5-20Kbp inserts– Genomic DNA or cDNAs

• Cosmids– 20-50Kbp genomic DNA inserts

• BAC (Bacterial Artificial Chromosomes)– 100-500Kbp genomic DNA sequences

• YAC (Yeast Artificial Chromosomes)– 100Kbp – 1Mbp genomic DNA sequences

Libraries – Sources of Genes for Cloning

• Genomic libraries – genomic DNA (coding & non-coding) inserted into as

RE fragments into a vector– Each vector contains a single small piece of the genome– Bazillions of vectors are present in the library so that all

of the genomic DNA is represented in at least 1 vector.– The vectors are maintained in a host cell (bacteria or

yeast)

Creating a Library

• Genomic DNA or cDNAs are ligated into vectors

• Vectors are inserted into hosts– Transformation by plasmids– Infection by phage

• Each host contains 1 copy of a particular recombinant DNA

• Each host replicates and produces many clones of that recDNA sequence

Generation of Genomic Library

RE sites

Libraries - Sources of Genes for Cloning

• cDNA libraries– cDNA = complementary DNA – cDNA is made from mRNA– cDNAs represents the genes actually being expressed as

mRNAs• cDNA can be made from particular cells, tissues,

embryonic stages, etc• most useful library for identifying differentially expressed

genes• cDNA must be used because mRNA can not be

manipulated like DNA – ie ligated into a vector and replicated

Figure 16.8 Synthesizing Complementary DNA

Getting New Genes into Cells

• When DNA is introduced to a population of host cells only a few cells incorporate and express it (transformants).

• Only a few vectors that move into cells actually contain the new DNA sequence (insert)

• A method for selecting for transformants and screening for inserts is needed.

• Commonly used approach to this problem is illustrated using E. coli as hosts, and a plasmid vector with genes for antibiotics resistance

Identifying Recombinant Transformants

• 3 possible products of ligation reactions– Self-religated vector– Self-ligated insert– Recombinant vector (vector + insert)

• Transformation of mixture into host gives– Non-transformant – no DNA of any kind taken up– Transformants – some type of DNA taken-up– Recombinants – take up recombinant vector

•

Selection of Transformants

• Hosts are chosen that are sensitive to a particular substance or require a particular nutrient (auxotrophs)

• The vector provides the genes needed to be resistant to the substance or produce the nutrient

• Host cells taking up vector or recombinant vector live• Host cells taking up insert or no DNA at all die• Transformants live – are selected for automatically• Non-transformants die – are selected against

Identify Recombinants by Screening• Transformants with a recombinant vector may express or

fail to express a particular gene product– LacZ

• Made by non-recombinants• Not made by recombinants• Turns cells blue in presence of a special subsrate

• Transformants with vector alone are distinguishable from those with a recombinant vector

Using Cloned Genes

• Study expression pattern of genes to gain insight to function

• Sequence comparison of related genes – allows determination of important protein functional domains– infers evolutionary relationships

• Site-directed mutagenesis – specific alteration of DNA sequence (ergo protein sequence) to

study protein function • Genetic manipulation

– gene knockouts & knockdowns

• Production of recombinant proteins– insulin, TPA– Biotechnology Realm

Figure 16.13 An Expression Vector Allows a Foreign Gene to Be Expressed in a Host Cell

Figure 16.14 Tissue Plasminogen Activator: From Protein to Gene to Drug

Using Cloned DNA – Genetic & Biomedical Technologies

• Targeted Gene Disruption – Knockouts & knockdowns

• Homologous recombination (knockouts)– replacement of endogenous gene with mutated version in

genome– insight to role of gene during development or disease

• RNA interference (knockdowns)– expression of antisense RNA – expression or injection of small inhibitory RNA (siRNA)– block translation of mRNAs ergo loss of gene expression at

protein level

Figure 16.9 Making a Knockout Mouse (Part 1)

Figure 16.9 Making a Knockout Mouse (Part 2)

Figure 16.11 Using Antisense RNA and RNAi to Block Translation of mRNA

Studying Differential Gene Expression

• Genomics – large number of genes in eukaryotic genomes– distinctive pattern of gene expression in different

tissues at different times• To find patterns of gene expression

– DNA sequences arranged in an array - DNA chip– chip (array) is hybridized to cDNA or mRNA from

different sources

Figure 16.10 DNA on a Chip

DNA Chips

• Can be used to detect genetic variants • Diagnose human genetic diseases

– Chips contain oligonucleotides with possible mutant sequences

– Hybridization of patient DNA indicates what mutation they have (or if normal)

GMOs

• Selective breeding – used for centuries to improve species to meet human

needs.• Trans-Genetically Modified Organisms• Molecular biology is accelerating progress in these

applications. • Major advantages over traditional techniques:

– Specific genes can be affected.

– Genes can be introduced from other organisms.

– Plants can be regenerated much more quickly by cloning than by traditional breeding.

Biotechnology: Applications of DNA Manipulation• With the exception of identical twins, each human being

is genetically distinct from all other human beings.• Characterization of an individual by DNA base

sequences is called DNA fingerprinting.

Biotechnology: Applications of DNA Manipulation• Scientists look for DNA sequences that are highly

polymorphic.• Sequences called VNTRs (variable number of tandem

repeats) are easily detectable if they are between two restriction enzyme recognition sites.

• Different individuals have different numbers of repeats. Each gets two sequences of repeats, one from the mother and one from the father.

• Using PCR and gel electrophoresis, patterns for each individual can be determined.

Figure 16.17 DNA Fingerprinting

Biotechnology: Applications of DNA Manipulation• The many applications of DNA fingerprinting include

forensics and cases of contested paternity.• DNA from a single cell is sufficient to determine the

DNA fingerprint because PCR can amplify a tiny amount of DNA in a few hours.

• PCR is used in diagnosing infections in which the infectious agent is present in small amounts.

• Genetic diseases such as sickle-cell anemia are now diagnosable before they manifest themselves.