19.

1 Techniques of Molecular Genetics

Have Revolutionized Biology

• Recombinant DNA Technology—Genetic

Engineering—Biotechnology:

• Locating, isolating, altering, and studying DNA

segments

• Biotechnology:

• Using recombinant DNA technology to

develop new biological products
19.2 Molecular Techniques Are Used to
Isolate, Recombine, and Amplify Genes
 Cutting and Joining DNA Fragments

• Restriction enzymes: recognizing and cutting

DNA at specific nucleotide sequences
– Palindromic sequences
– Immune system of bacteria

• Type II restriction enzyme: most useful enzyme

• By adding methyl groups to the recognition

sequence to protect itself from being digested by
its own enzyme in bacteria
 Cutting and Joining DNA Fragments

• Cohesive ends: fragments with short, single-

stranded overhanging ends

• Blunt ends: even-length ends from both single

strands
 Viewing DNA Fragments

• Gel electrophoresis-separation of DNA

fragments by size through a gel medium
• Smaller fragments migrate faster
 Viewing DNA Fragments

• Locating DNA fragments with Southern

blotting and probes

• Probe: DNA or RNA with a base sequence

complementary to a sequence in the gene of
interest
• Is usually labeled for easy detection
• Radioactive P32
• Fluorescent tag
 Cloning Genes

• Gene cloning: amplifying a specific piece of

DNA via a bacteria cell

• Cloning vector: a replicating DNA molecule

attached with a foreign DNA fragment to be
introduced into a cell
– Has features that make it easier to insert DNA and
select for presence of vector in cell.
• Origin of replication
• Antibiotic resistance gene
• Cloning site
 Cloning Genes

• Plasmid vectors

• Linkers: synthetic DNA fragments containing

restriction sites

• Transformation of host cells with plasmids

• Selectable markers are used to confirm whether

the cells have been transformed or not.
 Amplifying DNA fragments with the
polymerase chain reaction (PCR)

• Taq polymerase: stable DNA polymerase at

high temperature
• Researcher designs specific oligonucleotide
primers that serve as the ends of the amplified
fragment
• Very similar to replication
19.3 Molecular Techniques Can Be Used
to Find Genes of Interest
 Gene Libraries

• Gene library: a collection of clones containing

all the DNA fragments from one source

• Creating a genomic DNA library

• cDNA library: consisting only of those DNA

sequences that are transcribed into mRNA

• Creating a cDNA library

 Gene Libraries

• Screening DNA libraries

• Plating clones of the library

• Probing plated colonies or plaques

 In Situ Hybridization

• DNA probes used to determine the

chromosomal location and to visualize a gene
while it is in a cell
• May also be used to detect localization of mRNA
expression in multicellular organisms.
• Positional Cloning
• Isolating genes on the basis of their position
on a genetic map
19.4 DNA Sequences Can Be Determined
and Analyzed

• Restriction Fragment Length Polymorphisms

(RFLPs)

• Some DNA fragments have different

restriction sites due to mutation for the same
restriction enzyme, which causes
polymorphisms within a population.
 DNA Sequencing

• Sanger’s dideoxy-sequencing method

• Dideoxyribonucleoside triphosphate (ddNTP)

lacks a 3′-oh group, which terminates DNA
synthesis.
 DNA Fingerprinting (DNA Profiling)

• Microsatellites: variable number of copies of

repeat sequences possessed by many
organisms, which can be amplified by PCR

Combined with RFLP analysis to form more

thorough fingerprint
19.5 Molecular Techniques Are Increasingly
Used to Analyze Gene Function

• Forward genetics:
– Begins with a phenotype to a gene that
encodes the phenotype

• Reverse genetics:
– Begins with a gene of unknown function, first
inducing mutations and then checking the
effect of the mutation on the phenotype
19.5 Molecular Techniques Are Increasingly
Used to Analyze Gene Function

• Site-directed mutagenesis

• Reverse genetics: creating mutation in

particular DNA sequences, and then studying
the effects of these mutation on the
organisms
19.5 Molecular Techniques Are Increasingly
Used to Analyze Gene Function

• Silencing genes with RNAi

• Using RNAi for the treatment of human

disease: lowering ApoB with RNAi

• Model Genetic Organism

• The mouse, Mus musculus

19.6 Biotechnology Harnesses the Power
of Molecular Genetics

• Pharmaceuticals
• Human insulin
• Specialized bacteria
• Agricultural products
• Oligo nucleotide drugs
• Genetic testing
• Gene therapy
• Direct transfer of genes into humans to
treat disease