Virginia Bugeja Recombinant DNA Technology Lecture 1

Learning Outcomes:
To provide an introduction to the topic
To begin to explore basic steps in recombinant DNA technology

What are the 2 TYPES OF GENE AMPLIFICATION?

Cell-based cloning
In vitro amplification

What is CELL-BASED CLONING?

The introduction of manipulated genetic material into a cell in such a way
as to allow it to replicate and be passed to progeny cells

What are the 2 TYPES OF IN VITRO AMPLIFICATION?

Polymerase Chain Reaction (PCR) DNA
Reverse Transcriptase PCR (RT-PCR) RNA

Virginia Bugeja Recombinant DNA Technology Lecture 2

Learning Outcomes:
To understand the principles of DNA isolation
To understand the properties and use of restriction enzymes for cutting
DNA

What ENZYME IS USED TO CUT DNA?

Restriction endonuclease

What are the RECOGNITION SPECIFIC SEQUENCES?

Palindromic

What are the 2 TYPES OF CUTS MADE BY RESTRICTION ENDONUCLEASE?

Asymmetrical cuts sticky ends
Symmetrical cuts blunt ends

How is the COMMON STAIN USED TO STAIN GELS?

Ethidium bromide

What TYPE OF AGENT IS ETHIDIUM BROMIDE?

Intercalating agent

What is the ALTERNATIVE TYPE OF STAIN?

GelRed

How is the GEL VIEWED?

UV light

What are the 2 WAYS SEPARATION OCCURS FOR NUCLEIC ACID

ELECTROPHORESIS?
Linear molecules according to size
Conformation (shape)

Virginia Bugeja Recombinant DNA Technology Lecture 3

Learning Outcomes:
To understand the molecular techniques for DNA analysis
To understand the principles of DNA hybridisation
To understand the concept of a nucleic acid probe

What TYPE OF BLOTTING IS USED TO INVESTIGATE DNA?

Southern blotting

What TYPE OF MEMBRANE IS USED DURING BLOTTING?

Nylon

What must happen to the DNA BEFORE TRANSFER ONTO NYLON

MEMBRANE?
DNA must be denatured

What TYPE OF SOLUTION IS THE GEL SOAKED IN?

Alkali NaOH

What TYPE OF PROBE IS USED FOR SOUTHERN BLOTTING?

Single stranded DNA

What CHARGE IS THE NYLON HYBRIDISATION MEMBRANE?

Positive charge

What is HYBRIDISATION USED FOR?

Identify similar DNA sequences

What TYPE OF BLOTTING IS USED TO INVESTIGATE RNA?

Northern blotting

What TYPE OF BLOTTING IS USED TO INVESTIGATE PROTEINS?

Western blotting

What TYPE OF PROBE IS USED FOR WESTERN BLOTTING?

Antibody

Virginia Bugeja Recombinant DNA Technology Lecture 4

Learning Outcomes:
 To understand the principles and applications of the polymerase chain
reaction (PCR)

What are the 4 REQUIREMENTS FOR PCR?

Primers
Oligonucleotides
Taq polymerase
Thermostable enzyme

What are the 3 STEPS OF PCR?

Denaturation
Annealing
Extension

What happens during DENATURATION?

DNA strands separate

What happens during ANNEALING?

Forward and reverse primers bind to specific complementary target
sequences

What happens during EXTENSION?

The polymerase extends from primers to create polymer

What are 3 APPLICATIONS OF PCR?

Medical diagnosis
Forensics
Molecular evolution

How many CYCLES ARE THERE IN PCR FOR MILLION-FOLD AMPLIFICATION?

30

Virginia Bugeja Recombinant DNA Technology Lecture 5

Learning Outcomes:
 To understand the processes of ligation and transformation
To understand the concept of a vector as used in DNA cloning
To understand the concept of an expression vector as used in DNA
cloning

What are the 2 STEPS OF MAKING RECOMBINANT DNA?

Combine DNA fragments with complementary ends
Add DNA ligase

What does LIGATION REACTION LEAD TO?

Covalent linkage of DNA fragments

What TYPE OF CELLS DOES TRANSFORMATION REQUIRE?

Competent cells

What are the 2 METHODS OF MAKING CELLS COMPETENT?

Heat shock + CaCl2
Electroporation

What are VECTORS?

Carrier molecules which allow the replication of DNA in living cells

What are the 2 EXAMPLES OF VECTORS?

Plasmids
Phage DNA

What are the 4 PROTEIN EXPRESSION SYSTEMS?

Bacterial
Viral
Eukaryotic
In vitro transcription/translation

Virginia Bugeja Recombinant DNA Technology Lecture 6

Learning Outcomes:
 To understand the principles and applications of nucleic acid
hybridisation
To understand aspects of gene analysis

What is HYBRIDISATION USED TO IDENTIFY?

Similar DNA sequences

What does HYBRIDISATION OCCUR BETWEEN?

2 single strands of nucleic acid

What 3 FACTORS INFLUENCE HYBRIDISATION?

Temperature
Salt conditions
Denaturing additives

What are 3 PROBE CHARACTERISTICS AFFECTING HYBRIDISATION?

GC-richness
Length
Similarity to target

What can NUCLEIC ACID HYBRIDISATION BE USED TO OBTAIN?

Specific gene of interest

What is a GENOMIC LIBRARY?

A collection of DNA clones that together carry a representative copy of
every DNA sequence in the genome of the particular organism

What is a CDNA LIBRARY?

A collection of cDNA clones that are representative of the mRNAs
expressed by a particular cell type, tissue, organ or organism

What can NUCLEIC ACID HYBRIDISATION BE USED IN?

Gene analysis

Virginia Bugeja Mutations Lecture 1

Learning Outcomes:
Chromosomal mutations
 Gene mutations
Biological consequences of mutation

What are MUTATIONS?

Heritable changes in DNA base sequences

What are the 2 CLASSES OF MUTATIONS?

Somatic
Germline

What are SOMATIC MUTATIONS?

Can be restricted to one individual

What are GERMLINE MUTATIONS?

Can affect many generations of individuals

What are the 2 TYPES OF MUTATIONS?

Spontaneous
Induced

What are the 2 DIFFERENT LEVELS WHICH MUTATIONS CAN OCCUR AT?
Chromosomal level
Base pair level

What are CHROMOSOMAL MUTATIONS?

Changes in chromosome structure

What are the 4 TYPES OF CHROMOSOMAL MUTATIONS?

Deletions
Duplications
Inversions
Translocation

What are BASE PAIR MUTATIONS?

Changes in gene structure

What are the 4 TYPES OF BASE PAIR MUTATIONS?

Point
Insertion
Deletion
Inversion

What are the POINT MUTATIONS?

Single base mutations

What are POINT MUTATIONS RECOGNISED AS?

Single nucleotide polymorphism (SNPs)
What are the 2 CLASSES OF POINT MUTATIONS?
Transition
Transversion

What is WILD-TYPE?
Most frequent allele observed in a population

What is MUTANT?
Rare allele observed in a population

What is PHENOTYPE?
Observable characteristic

What is GENOTYPE?
Genetic sequence responsible for determining a phenotype

What are the 2 TYPES OF PYRIMIDINE NUCLEOTIDES?

T
C

What are the 2 TYPES OF PURINE NUCLEOTIDES?

G
A

What are TRANSITION MUTATIONS?

Interchange of bases of same shape
Alternative pyrimidines
C <-> T

What are TRANSVERSION MUTATIONS?

Interchange of bases of different shape
Purine <-> Pyrimidine
C <-> G

What are MOST MUTATIONS?

Transition mutations

What are EXAMPLES OF INSERTION MUTATIONS?

Transposable elements

What are the 4 CONSEQUENCES OF MUTATION ON ENCODED PROTEINS?

Silent mutation
Missense mutation
Nonsense/Chain termination mutation
Frameshift mutation

What is a SILENT MUTATION?

Do not change phenotype
What is a MISSENSE MUTATION?
Changes to a different amino acid

What is a NONSENSE/CHAIN TERMINATION MUTATION?

Changes to a stop codon

What is a FRAMESHIFT MUTATION?

Caused by indels that shifts the reading frame

Virginia Bugeja Mutations Lecture 2

Learning Outcomes:
Biological consequences of mutation
Forward and reverse mutation
Phenotypic consequences of mutations

What is a FORWARD/REVERSE MUTATION?

Wildtype Mutant
What are the 5 PHENOTYPIC CONSEQUENCES OF MUTATIONS?
Lethal
Conditional lethal
Biochemical
Morphological
Little effect

What is a LETHAL MUTATION?

The loss of an essential protein function

What is a CONDITIONAL LETHAL MUTATION?

Mutations in essential genes but protein activity is lost under certain
conditions

What is a BIOCHEMICAL MUTATION?

Mutants in metabolic pathways

What is an AUXOTROPH?
Cell or strain that requires additional substances for growth above those
required for the wild type

What is a PHOTOTROPH?
Cell or strain that has no nutritional requirements beyond those of the wild
type
Grows on minimal medium

What is a MORPHOLOGICAL MUTATION?

Visible mutations

What are the 2 TYPES OF MUTATIONS WITH LITTLE EFFECT?

Neutral
Silent

Virginia Bugeja Mutations Lecture 3

Learning Outcomes:
Causes of mutations
o Spontaneous mutation
o Induced mutation
Repair mechanisms
Ames test

What are the 2 TYPES OF CAUSES OF MUTATIONS?

Spontaneous
Induced

What is a SPONTANEOUS MUTATION?

Random changes in the nucleotide sequence

What is NOT INVOLVED IN SPONTANEOUS MUTATIONS?

Mutagens

What TYPE OF SHIFT CAN SPONTANEOUS REARRANGEMENT CAUSE?

Tautomeric

What can TAUTOMERIC SHIFTS RESULT IN?

Mismatched base pairs

What is an INDUCED MUTATION?

Caused by mutagens

What are the 3 TYPES OF MUTAGENS?

Chemical
Physical
Biological

What are 3 TYPES OF CHEMICAL MUTAGENS?

Direct modification of DNA bases
Base analogue
Intercalating agents

What are 3 TYPES OF PHYSICAL MUTAGENS?

Gamma
X-rays
UV light

What are the 2 TYPES OF REPAIR MECHANISMS?

Photoreactivation
Excision repair

What TEST IS USED TO DETERMINE WHETHER A CHEMICAL IS A MUTAGEN?

Ames test

What does the AMES TEST USE?

Auxotrophic mutants of bacteria
 Colin Malcolm Genetic Analysis Lecture 1

What was MENDELS LAW OF SEGREGATION?

One copy of each gene per parent is passed onto the progeny

What is USED TO SHOW ALL THE POSSIBLE COMBINATIONS OF ALLELES?

Punnett square

What are the 2 TYPES OF TRAITS?

Dominant
Recessive

What is CODOMINANCE?
Both alleles are expressed equally
What is POLYMORPHISM?
Multiple alleles

What is an EXAMPLE OF POLYMORPHISM?

ABO blood groups

What is PLEIOTROPY?
1 allele affects more than 1 trait

What is an EXAMPLE OF PLEIOTROPY?

Sickle cell anaemia shows resistance to malaria

What is EPISTASIS?
A genes presence suppresses the effect of a gene at another locus

What is PENETRANCE?
The proportion of individuals with the same genotype that show the same
phenotype

What is EXPRESSIVITY?
The degree that a genotype is expressed in a phenotype

Colin Malcolm Genetic Analysis Lecture 2

What is MITOSIS?
One cell division that produces 2 daughter cells identical to parent cell

What PHASE IS CHROMOSOMAL REPLICATION INVOLVING DNA SYNTHESIS

WHICH OCCURS PRIOR TO CELL DIVISION?
S phase

What does REPLICATION PRODUCE?

Pair of identical sister chromatids

What does SYNAPSIS DEPEND ON?

Synaptonemal complex
What is the JOINING OF HOMOLOGOUS PAIRS OF CHROMOSOMES CALLED?
Synapsis

What are REPLICATE SISTER CHROMATIDS CALLED?

Dyad

What is a PAIR OF SYNAPSED DYADS CALLED?

Bivalent

What are 4 CHROMATIDS IN A BIVALENT CALLED?

Tetrad

What is formed when 2 DYADS FORM A BIVALENT?

Chiasma

What term is given when 2 ALLELES ARE LOCATED ON THE SAME

HOMOLOG?
In coupling/cis conformation

What term is given when 2 ALLELES ARE LOCATED ON DIFFERENT

HOMOLOGS?
Uncoupled/trans conformation

What SYMBOL IS USED FOR ALLELE ON THE SAME HOMOLOG?

No symbol

What SYMBOL IS USED FOR ALLELE ON DIFFERENT HOMOLOGS?

Semicolon

What is the EQUIVALENT OF 1% RECOMBINANTS IN MAP UNIT AND

CENTIMORGAN (CM)?
1

What is the RECOMBINANT PERCENTAGE FOR LINKED GENES?

Less than 50%
What is the RECOMBINANT PERCENTAGE FOR UNLINKED GENES?
50% and more

How is MAP DISTANCE DETERMINED?

Number of crossovers