DNA Technology

DNA technology uses: Genetic engineering Genetically modified (GM) crops Gene Therapy Genetic (DNA) Fingerprinting Genetic Screening Personalised medicine

Techniques used in Dna tech Amplifying DNA: PCR Comparing DNA: identifying and using specific marker sites using restriction endonuclease enzymes & DNA probes

Polymerase Chain Reaction

PCR is used in: forensic science e.g. genetic fingerprinting; medical research e.g. genetic screening to find alleles for genetic diseases; scientific research e.g. to investigate DNA of extinct species to determine their relationship with living organisms; The Human Genome Project. Its purpose is to amplify very small quantities of DNA producing, many identical copies of the selected section of DNA in a very short time.

method Heat to 95oC to break hydrogen bonds between complementary bases and separate strands Cool to 40-60oC & add excess primer Primers are short single stranded sequences of about 20 nucleotides which are complementary to bases in part of the DNA strand being copied. Cooling allows the primers to bind (anneal) to each DNA strand at specific complementary points Primers stop the DNA strands rejoining They bracket the DNA to be copied by attaching to the end of the DNA sequence and shows DNA polymerase where to start copying. There are 2 different primers as there are 2 different ends of the DNA DNA replication can only start with a double stranded region

Heat to 70oC. Add free DNA nucleotides & thermostable DNA polymerase Polymerase copies each strand , starting with the primers. The polymerase is thermostable allowing the process to occur rapidly at the higher temperatures required, without denaturing. The enzyme was originally isolated from a bacterium that lives in hot springs. TWO DNA molecules will be formed following PCR and are used as templates as the process is repeated. This allows millions of copies of the original DNA to be produced in a very short time.

HEAT 95oC HEAT 70oC Cool 40-60oC primer DNA polymerase primer Heat causes Hydrogen bonds between bases to break. Heat acting in same way as DNA polymerase during replication.

key requirement AVOID CONTAMINATION with any other DNA source as both the original & contaminated DNA will be amplified Double Helix heated, unwinds 9

The Polymerase Chain Reaction sumanisinc brill animation http://www.maxanim.com/genetics/PCR/pcr.swf www.dnalc.org/shockwave/pcranwhole.html/southan.html

that are used to identify specific DNA sequences genetic markers sections of DNA that are used to identify specific DNA sequences Allows for comparison of DNA sequences between different people

restriction endonuclease enzymes Enzymes that recognise & cut specific nucleotide sequences. Originally from bacteria that use RE enzymes to cut DNA of attacking bacteriophages.

produces sticky ends because G & A don’t lie opposite each other. e.g. EcoR1 recognises the sequence GAATTC, cutting between the G & A of each strand produces sticky ends because G & A don’t lie opposite each other.

3’ CCCTAGG 5’ GGGATCCTTAA 3’ CCCTAGGAATTCAGACC 5’ AATTCAGACC 5’ GTCTGG 3’ 5’ GGGATCCTTAAGTCTGG 3’ sticky ends

e.g. Bam1 recognises the sequence GGATCC, cutting between the G & G of each strand produces sticky ends because G & G don’t lie opposite each other. …G G A T C C… …C C T A G G…

e.g. Hae 111 recognises the sequence GGCC, cutting between the G & C of each strand produces blunt ends because G & C lie opposite each other, these are not as useful as sticky ends. …G G C C… …C C G G…

Restriction enzymes will cut DNA into a number of fragments, depending upon the number of recognition sites in the sample of DNA. The size of the fragment depends on how far apart the recognition sites are. If DNA has 4 recognition sites, 5 fragment lengths of DNA are produced. Recognition site 1 Recognition site 2 Recognition site 3 Recognition site 4 DNA SAMPLE 5 fragments

If two samples of DNA are identical, treatment with a particular restriction enzyme will produce the same number of DNA fragments, each of similar size. However, different individuals have different DNA sequences and therefore produce different fragments when a particular restriction enzyme is used to cut an equivalent section of DNA. The fragments produced are called RFLPs, RESTRICTION FRAGMENT LENGTH POLYMORPHISMS.

This allows for comparison or identification of DNA samples PERSON A Recognition site 1 Recognition site 2 Recognition site 3 Recognition site 4 DNA SAMPLE 5 fragments PERSON B Recognition site 1 Recognition site 2 Recognition site 3 Recognition site 4 Recognition site 5 DNA SAMPLE 6 fragments This allows for comparison or identification of DNA samples

Restriction Fragment Length Polymorphisms RFLPS pronounced RiFlips Restriction Fragment Length Polymorphisms http://highered.mcgraw-hill.com/olc/dl/120078/bio20.swf

reasons why different people have different DNA sequences SNPs MRSs

Single Nucleotide Polymorphisms snps pronounced snips Single Nucleotide Polymorphisms

A SNP is …… a difference in a single nucleotide in a section of DNA It can give rise to different alleles for a gene egs include sickle cell anaemia (chromosome 11)

Sickle cell anaemia Thr Pro Glu Glu Glu ...ACT CCT GAG GAG GAG... Thr normal Hb chain Amino acid sequence Thr Pro Glu Glu Glu DNA base sequence ...ACT CCT GAG GAG GAG... codon number 4 5 6 7 sickle cell Hb chain Amino acid sequence Thr Pro Val Val Glu DNA base sequence ...ACT CCT GTG GTG GAG...

Chromosome 16 Genetically Ginger RSci Advent gcsbio.weebly.com

Microsatellite Repeat Sequences MRSs Microsatellite Repeat Sequences (Also called variable number tandem repeats – VNTRs).

Only approximately 2% DNA codes for proteins and 3% has a regulatory role. The remaining 95% has unknown function and is called non-coding DNA. Within this DNA there are many sections where a small number of bases are repeated many times e.g. ….ATCATCATCATCATCATCATC…

These sequences are called microsatellite repeat sequences or short tandem repeats. The number of repeats of these MRSs are unique to an individual.

Short sequences of single stranded DNA dna PROBE Short sequences of single stranded DNA with known nucleotide and base sequence. May be fluorescent or radioactively labelled, to enable it to be located. Radioactive probes contain radioactive isotopes of nitrogen in their nucleotide structure.

The DNA is treated to separate the DNA strands (heat) Add probe – it base pairs with complementary DNA sequence. Detect the target sequence: Fluorescent probe use a laser scanner Radioactive probe use X-ray film

They are used to identify specific gene sequences . Many inherited diseases are caused by a single allele with a known base sequences A probe complementary to the allele nucleotide sequence is used: If it binds the disease is present If it doesn’t bind it is not present.

Pre implantation genetic diagnosis PGD Pre implantation genetic diagnosis This is the use of DNA probes to detect defective genes in an embryo to reduce the chance of a hereditary disease in a family.

Measuring genetic variation allows the following questions to be answered: Does a particular gene differ from person to person? Are two persons closely related? Are certain alleles associated with an hereditary disease? How do genes vary within a population? How much genetic diversity is there within a particular species? How do genes differ from species to species? How can this be used to unravel the taxonomic relationship among species?

Genetic (dna) fingerprinting Extract DNA from a sample e.g. from a crime scene, often PCR required to amplify the DNA 2. Restriction Enzymes cut the DNA into different sized fragments Specific MRSs used as they can be measured more accurately than RFLPs. Primers used to flank the MRS region involved to ensure fragments only come from that region

3. DNA fragments separated using gel electrophoresis. DNA is placed into separate wells in agar gel. An electric current is applied across the length of the gel, with the negative electrode closest to the wells of DNA. DNA has a negative charge, so moves through the spaces in the gel towards the positive electrode. Smaller DNA fragments move further than larger fragments. 4. DNA is heated to separate the DNA strands to make it single stranded.

The gel will dry up, so the DNA fragments are transferred to a nylon membrane (process called Southern blotting) Radioactive or fluorescent DNA probes are added to the membrane and will join to the complementary base sequences in the microsatellite region selected. The sample is washed to remove any probes that do not base pair. The labelled DNA is added to X-ray film or a laser scanner is used to detect the radioactive or fluorescent probes and a pattern of bars appear, revealing the position of the DNA fragments: a DNA fingerprint. Diagram p82

definitions PROBES GENETIC MARKERS SNPS RFLPS MRSs