Genetic lab techniques

Cutting DNA

→ restriction digestion

• DNA can be cut by restriction endonucleases that recognise specific sequences

• Usually extracted from bacteria

• Restriction maps say where different enzymes will cut

E.g. Bsa2600 = 2600 bp

Nucleic acid hybridisation Basics

→ two single stranded nucleic acids (DNA or RNA) are allowed to interact so that hybrids form

• the DNA added is called a probe, and will only anneal to the corresponding sequence

Probes can be tagged with:

• radioactivity

• Fluorescence

Nucleic acid hybridisation method

1. Extract DNA, cut using restriction enzymes

2. Separate by size using electrophoresis

3. Transfer DNA to a nylon membrane

4. Radioactive DNA probe added to membrane (binds to specific fragments)

5. Membrane onto photographic paper

6. Radioactivity transferred

*fluorescence is used more now

Detection of chromosome translocation FISH

→ fluorescent in-situ hybridisation

• detecting prescience/absence of specific DNA sequences

• Utilities short fluorescents labelled DNA probes

• Used in genetic counselling, cancer etc

To identify chromosome translocations = where a segment from one chromosome is transferred to another

FISH example SRY

SRY = gene on Y chromosome that determines sex

• green = probe for part of X

• Orange = probe for SRY gene

Shows two X chromosomes in a male, with the SRY translocated onto it = present as male

Gel electrophoresis

• DNA fragments are separated into different lengths

• DNA is negative so moves towards the positive electrode in a gel

• Smaller fragments move faster through the gel

• DNA binding dye added and detected using UV

• Bands are seen and can identify no. Of base pairs in each band by how far they travel

Agarose = low resolution - large fragments

Polyacrylamide = high resolution - short fragments

DNA synthesis in vivo ingredients

In vivo = organism

• DNA polymerase

• DNTPs

• Template DNA

• Primer

DNA synthesis in vitro ingredients

In vitro = test tube

• DNA polymerase

• DNTPs

• Template DNA

• Synthetic primer (oligonucleotides)

• Buffer (Mg2+)

PCR basics and ingredients

→ used to amplify large quantities of a specific sequence of DNA from a small sample

• each cycle doubles the amount - 30 cycles = 20 (index 30)

• PCR products include the primers

• template DNA

• Primers (2)

• DNTPs

• Buffer Mg2+

• Taq polymerase

PCR method

1. Desaturation = double DNA strand melts open 95*C

2. Annealing = (2) primers bind to DNA and polymerase attaches and starts copying about 50*C

3. Extension = DNA polymerase extends from annealed primer at 72*C

Amplification of STRs

STR = simple tandem repeats

Amplify STRs from small bits of DNA in crime scenes

• 2-6 base pairs of DNA

• Higher mutation rate than most sequences

• Uses PCR to amplify, but one primer is fluorescently labelled

• Electrophoresis then takes place in a thin capillary with polyacrylamide

• Lasers detect exact size

Transgenics

→ movement of DNA between species

• genetic code is universal so expression is possible

GM - molecular cloning

→ set of lab techniques that allow us to insert a fragment of foreign DNA into a vector to create RECOMBINANT DNA that is capable of replicating

Types of vectors:

• plasmid - cloning of small fragments

• Yeast artificial chromosome - large fragments

Strands joined together by DNA ligaments to form phosphodiester bonds

Amplifying recombinant DNA

Bacterial host- most common E.coli

Eukaryotic host = yeast

• cleave DNA and create recombinant

• Introduce into bacterial cell

• Cell culture produces millions of bacteria

• Many copies of purified plasmid isolated

Artificial plasmid purification

• obtain an ‘origin of replicaion’ and a ‘cloning site’

• Contain a ‘selectable marker’ (usually an antibiotic resistance gene)

• Bacterial cells then exposed to an antibiotic = only those with plasmid survive

= purified

Artificial plasmid areas

How can DNA be expressed in cells

Gene library

→ collection of recombinant clones

E.g. each bacteria has a small part of DNA from the fly inserted into a plasmid

• can then screen for clones containing genes of interest by DNA/RNA hybridisation or DNA sequencing

Sickle cell mutation though functional cloning

• isolated mRNA from blood of SC patient

• Used reverse transcriptase to make cDNA from mRNA

• Insert cdNA into plasmids then transform bacteria to make a cDNA library

• Used an antibody for haemoglobin to identify bacterial colonies containing the gene for haemoglobin (inserted into a plasmid)

• Found that sickle cell haemoglobin is the result of a single base pair mutation