Biosci101: Genetics

Recombinant Genes

Genes from two different sources that are combined in vitro to form the same molecule

In vitro

In a test tube

In Vivo

In organism

Biotechnology

The manipulation of organisms or their components to make useful products

Genetic Engineering

A subset of Biotechnology

The In-Vitro alteration or recombination of genetic material (DNA or RNA) and the reintroduction of the altered genetic material into a living organism

Genetic Engineering vs Selective Breeding

Precise choice of 1 or few genes --- New combination of many genes

Genes from any species --- genes only from related species

Gene expression can be controlled --- No control (via promoters)

Restriction Enzymes

Naturally occurring in bacteria. Cut DNA at specific 4-8bp sections

Natural use of restriction enzymes in bacteria

defence mechanism against bacteria (prevent introduction of DNA from phages)

Example of a restriction enzyme

EcoRI- this cuts at GAATTC

Meaning of Palindromic Restriction Sites

The sequence is the same when reversed and complementary

Most Restriction sites are like this

Restriction enzymes that leave blunt ends

sticky end or blunt end cutters

DNA Ligase

Covalently bonds sugar phosphate backbone of nucleotides back together

Cloning Vector

Any molecule that can carry foreign DNA

eg: bacteriophages / plasmids

Plamids

Circular DNA that is autonomously replicated inside host bacterial cell

(easily isolated from host)

Host Bacteria

Gene is inserted into these from the vector

Transformation

Method of introducing a plasmid of DNA into bacterial cells

A cell is competent when ____

'it is ready to take up a plasmid

How to make bacterial cells competent

Electroporation

Calcium and Heat shock treatment

Two types of Selectable Markers

To identify if the bacteria is transformed

Antibiotic resistance plasmid

To identify if the plasmid used was recombinant

marker that changes colour (use of X-Gal that is broken down by lac-gene to release indigo dye)

3 Steps of Polymerase Chain Reaction

1) Denaturing

2) Annealing

3) Extension

Denaturing

Heating of the sample to 90C to separate two DNA strands

Annealing

Allowing cooling of sample (50-60C)

Primers are also added to prevent strands rebinding

Length of Primers

18-25bp

Why are primers in excess for annealing

so that the cooling DNA strands are more likely to bind to primers that other DNA

Extension

In PCR this is the process by which polymerase adds new nucleotides to the chain

How the only the desired region is obtained in PCR

Due to primers binding at the ends of the regions by the end of the 3rd cycle there will be 2 molecules of only the target sequence

By cycle 30 there are >1,000,000 of the desired molecule

Factors that affect rate of movement in Gel Electrophoresis

Size (main form of separation for DNA)

Electrical Charge

etc.

Substance used for matrix in DNA Gel Electrophoresis

Agarose

How are electropherograms calibrated to read length of DNA at each band

Size markers using DNA of specific known lengths

The Genome

The total compliment of DNA that makes up inherited genetic material of an organism

Why do Eukaryotes have a larger genome than Prokaryotes

Repetitive regions like transposons

Exons make up ___% of the genome

1.5%

The Transcriptome

The part of the genome that has been transcribed in to RNA

Refers to the RNA from different tissues (or same in different periods) to study changes in gene expression

Sanger method is also known as...

Dideoxy Chain termination Method

What is next generation sequencing

involves the sequencing of multiple strands of DNA simultaneously

Reproduction

When an entity undergoes division that results in tHe production of two entities of the same kind

At what stage of replication are centrosomes paired

G2 of Interphase

What happens in Prophase

Mitotic spindle begins to appear

Chromosomes appear and become paired while connected at centromere

What happens in Prometaphase

Nuclear envelope fragments

Mocrofibre Spindle fibre connects to kinetochore

Kinetochore

Protein component of centromere that spindle fibres attach to

What happens in Metaphase

Chromosomes line up along metaphase plate

What happens in anaphase

Chromatids/chromasomes pull apart

What happens in in telophase and cytokinesis

Cleavage furrow forms

Nucleolus and nuclear envelope reforms

Meiosis

The two successive nuclear division in which a single diploid cell forms four haploid nuclei

Important events in Prophase I

Homologous chromosomes pair up

Chiasmata form and crossing over takes place

Tetrad

Structure consisting of 2 homologous paired chromosomes

Important events in Metaphase I

tetrads line up along metaphase plate

Important events in Anaphase I

Homologous chromosomes pulled apart

Independent assortment

The manner in which homologous chromosomes can align in a random orientation along the metaphase plate

Crossing Over

When similar regions of non-sister chromosomes can swap places to form recombinant chromosomes

Synapsis

The pairing up of homologous chromosomes in Prophase I

Sources of genetic Variation

Mutation (rare)

Crossing over

Independent Assortment

Random Fertilization

Heterochromatin

repetitive sequences of DNA that are not arranged into genes

Phenotype

Determined by interaction of alleles of the same (or different) genes and the influence of the environment to give an outward trait

Types of interactions of alleles

Complete Dominance

Incomplete (partial) dominance

Co-Dominance

Complete dominance

An allele is dominant if the phenotype is the same in both heterozygous and one of the homozygous forms

Incomplete Dominance

When the heterozygote shows an intermediate phenotype

Biological reason for Complete Dominance

One of the alleles results in the formation of a non-functional polypeptide (often the recessive)

Biological reason for Incomplete dominance

Due to the Dosage Effect

Dominant Homozygous gives double dose

Recessive homozygous - gene does not give dose (or lesser dose)

Heterozygous: results from the full dose of one and the partial dose of the other to give a new phenotype

Co Dominance

When there is full expression of both alleles in heterozygotes

Polygenic Inheritance

A mode of inheritance where the additive effect of multiple genes determines a single phenotypic characteristic

Type of characteristic that is usually polygenic

Quantitative Characteristics like skin colour

Mechanism of phenotype of skin colour

Controlled by 3 genes: Aa, Bb and Cc

AABBCC is very dark, aabbcc is very light

AABbcc=AaBbCc (same number of "dark" genes)

Norm of Reaction

The phenotypic range of a given genotype in different environments

Phenotype with a norm with no range

ABO blood groups

When are norms of reaction the greatest

In polygenic expression characteristics

Why is it difficult to study inheritance in humans

20 year generation time

relatively few offspring

Breeding experiments are ethically unacceptable

Mendel's Laws

1st: Principle of Segregation

2nd: Principle of Independant Assortment

Principle of Segregation

Two alleles of a gene separate in meiotic gamete production. The sperm and egg provide one allele of each gene when they come together

Principle of Independent Assortment

Each pair of alleles assorts independently from other pairs during formation of gametes

Only for unlinked genes

Sex Linked Traits

When the allele is located on one of the sex chromosomes

Hemizygous

When only one instance of an allele is present (males with X-linked traits)

Linked Genes

When alleles of different genes are located on the same chromosome so are inherited together (not assorted independently)

Outcome in offspring genotype with linked genes

No recombinant genotypes for the linked genes

How can there be recombinants with linked genes

If crossing over takes place (recombinants will be present in very low frequency)

Recombination rate of unlinked genes (different chromosome or vary far away on the same)

50% recombinantion

Recombination rate of linked genes

Less than 50%

Link Between recombination frequency and position of genes

Genes that are closer together are less likely to undergo crossing over

1 map unit =

1% recombination Frequency

What is the assumption made with map units

Rates of crossover will be constant along the chromosome

How to calculate recombinant frequqncy

Total number of recombinants

--------------------------------------------- x100

Total number of offspring

Reason that linkage maps will not be 100% correct

Assumption of constant crossover

Possibility of double crossover which gives phenotype of no crossover

How to reduce the error of double cross overs

Use more than 2 genes to create a linkage map

Heterogametic

The sex that produces two types of gametes and determined the sex in offspring

(males in humans - sperm with X or Y chromosome)

Homogametic

The sex that produces only one type of gametes

(females in humans)

Number of Chromosomes in Humans

46

22 pairs of autosomic and 2 sex chromosomes

Instances where sex in determined by the environment

Tuatara and alligator

Determined by temperature of incubation of the eggs

SRY Region

The sex determining region of Y chromosome that determines "Maleness"

Action of the SRY gene

Triggers testicular development

Pseudoautosomeal Regions

Regions of sex chromosomes that act somewhat autosomal

Androgen insensitivity Syndrome

Mutation where person does not respond to male hormones from testis so do not develop secondary male characteristic

Methods of Sex determination

X-O (one sex missing sex chromosome)

Z-W (similar system to humans)

Haplo-diplo system (one sex is haploid and the other diploid)

Reason for dosage compensation in females

due to presence of 2 X Chromosomes, X-linked genes would be expressed at twice the rate

When does X inactivation take place

around the 100 cell stage in development

Barr Body

An inactivated X chromosome

How are x chromosomes inactivated

Modification of DNA (addition of Methyl groups)

Modification of histones

Genes involved with X inactivation

XIST (X- Inactive Specific Transcript)

Action of XIST

RNA of XIST attaches to the X Chromosome and the interaction signals for further steps in the inactivation

Visible effects of X inactivation for Dosage compenstation

Tortoise shell female cats

Nondisjunction

When homologous chromosomes do not separate at either stage of meiotic metaphase